Recombinant modified vaccinia virus ankara (MVA) comprising human immunodeficiency virus (HIV) genes inserted into one or more intergenic regions (IGRs)

ABSTRACT

The invention relates to novel insertion sites useful for the integration of HIV DNA sequences into the MVA genome, and to the resulting recombinant MVA derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/355,948 filed Feb. 17, 2006, now U.S. Pat. No. 7,501,127 which is acontinuation-in-part of U.S. patent application Ser. No. 10/514,761,filed Nov. 16, 2004, now U.S. Pat. No. 7,550,147 which is the U.S.National Phase of International Application No. PCT/EP03/05045, filedMay 14, 2003. The contents of these applications are hereby incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to novel insertion sites useful for theintegration of HIV DNA sequences into the Modified Vaccinia Ankara(“MVA”) virus genome, and to the resulting recombinant MVA derivatives.

BACKGROUND OF THE INVENTION

Modified Vaccinia Ankara (MVA) virus is a member of the Orthopoxvirusfamily and has been generated by about 570 serial passages on chickenembryo fibroblasts of the Ankara strain of Vaccinia virus (CVA) (forreview see Mayr, A., et al., Infection 3, 6-14 [1975]). As a consequenceof these passages, the resulting MVA virus contains 31 kilobases lessgenomic information compared to CVA, and is highly host-cell restricted(Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). MVA ischaracterized by its extreme attenuation, namely, by a diminishedvirulence or infectious ability, but still holds an excellentimmunogenicity. When tested in a variety of animal models, MVA wasproven to be avirulent, even in immuno-suppressed individuals.

The Human Immunodeficiency virus (HIV) is the causative agent of theAcquired Immunodeficiency Syndrome (AIDS). Like all retroviruses, theHIV genome encodes the Gag, Pol and Env proteins. In addition, the HIVgenome encodes further regulatory proteins, for example, Tat and Rev, aswell as accessory proteins, such as Vpr, Vpx, Vpu, Vif and Nef.

Despite public health efforts to control the spread of the AIDSepidemic, the number of new infections is still increasing. The WorldHealth Organization estimated the global epidemic at 37.8 millioninfected individuals at the end of the year 2003, and 36.1 millioninfected individuals at the end of the year 2000, 50% higher than whatwas predicted on the basis of the data about a decade ago (WHO & UNAIDS.UNAIDS, 2004). Without further improvements on comprehensive preventionmechanisms, the number of new HIV infections to occur, globally, thisdecade is projected to be 45 million (2004 Report on The Global AIDSEpidemic, UNAIDS and WHO).

Given the steady spread of the epidemic, a number of different HIV-1vaccine delivery strategies, such as novel vectors or adjuvant systems,have now been developed and evaluated in different pre-clinicalsettings, as well as in clinical trials. The first vaccine candidatethat entered a phase-III clinical trial is based on envelope gp 120protein in alum formulations (Francis et al., AIDS Res. Hum.Retroviruses 14 (Suppl 3)(5): S325-31 [1998]). The results of the firstclinical studies were discouraging.

The viral vaccines that were tested for efficacy in the past are usuallybased on single HIV proteins, such as Env. However, even if an immuneresponse was generated against such a single protein, for example, Env,the immune response proved ineffective. One reason for theineffectiveness is the high mutation rate of HIV, in particular withrespect to the Env protein, reportedly resulting in viruses, in whichthe proteins are not recognized by the immune response induced by thevaccine. Since no effective prophylactic treatment is available, thereis still a need to bring an effective vaccine to the clinic.

Several excellent properties of the MVA strain pertinent to its use invaccine development have been demonstrated in extensive clinical trials(Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987]).During these studies, performed in over 120,000 humans, includinghigh-risk patients, no side effects were seen (Stickl et al., Dtsch.med. Wschr. 99, 2386-2392 [1974]).

It has been further found that MVA is blocked in the late stage of thevirus replication cycle in mammalian cells (Sutter, G. and Moss, B.,Proc. Natl. Acad. Sci. USA 89, 10847-10851 [1992]). Accordingly, MVAfully replicates its DNA, synthesizes early, intermediate, and late geneproducts, but is not able to assemble mature infectious virions, whichcould be released from an infected cell. For this reason, namely, itsreplication-restricted nature, MVA serves as a gene expression vector.

More recently, MVA was used to generate recombinant vaccines, expressingantigenic sequences inserted either at the site of the thymidine-kinase(tk) gene (U.S. Pat. No. 5,185,146), or at the site of a naturallyoccurring deletion within the MVA genome (PCT/EP96/02926).

However, although the tk insertion locus is widely used for thegeneration of recombinant poxviruses, particularly for the generation ofrecombinant Vaccinia viruses (Mackett, et al. P.N.A.S. USA 79, 7415-7419[1982]), use of this technology in MVA has several drawbacks. It wasreported by Scheiflinger et al. that MVA is much more sensitive tomodifications of the genome, when compared to other poxviruses, whichcan be used for the generation of recombinant poxviruses. Scheiflingeret al. reported, in particular, that one of the most commonly used sitesfor the integration of heterologous DNA into poxviral genomes, namely,the thymidine kinase (tk) gene locus, cannot be used to generate stablerecombinant MVA. Any resulting tk(−) recombinant MVA proved to be highlyunstable and, upon purification, immediately deleted the inserted DNA,together with parts of the genomic DNA of MVA (Scheiflinger et al., ArchVirol 141: pp 663-669 [1996]).

Instability and, thus, high probability of genomic recombination is aknown problem within pox virology, and a drawback for vaccineproduction. Actually, MVA was established during long-term passagesexploiting the fact that the viral genome of CVA is unstable whenpropagated in vitro in tissue cultured cells. Several thousands ofnucleotides (31 kb) had been deleted in the MVA genome, which,therefore, is characterized by 6 major deletions, and numerous smalldeletions, in comparison to the original CVA genome.

The genomic organization of the MVA genome has been described recently(Antoine et al., Virology 244, 365-396 [1998]). The 178 kb genome of MVAis densely packed and comprises 193 individual open reading frames(ORFs), which code for proteins of at least 63 amino acids in length. Incomparison with the highly infectious Variola virus, and also with theprototype of Vaccinia virus, namely the strain Copenhagen, the majorityof ORFs of MVA are fragmented or truncated (Antoine et al., Virology244, 365-396 [1998]). However, with very few exceptions, all ORFs,including the fragmented and truncated ORFs, get transcribed andtranslated into proteins. In the following description of the invention,the nomenclature of Antoine et al. (supra) is used and, whereappropriate, the nomenclature based on HindIII restriction enzyme digestis also indicated.

To date, only the insertion of exogenous DNA into the naturallyoccurring deletion sites of the MVA genome reportedly led to stablerecombinant MVAs (PCT/EP96/02926). Unfortunately, there are only arestricted number of naturally occurring deletion sites in the MVAgenome. Thus, a need exists for the identification of additional stableinsertion sites, particularly those that can be useful for generation ofMVA-based vaccines for treatment and/or prevention of AIDS.

SUMMARY OF THE INVENTION

Accordingly, this invention provides further insertion sites of the MVAgenome, and provides insertion vectors that direct the stable insertionof exogenous DNA sequences into these newly identified insertion sitesof the MVA genome.

Furthermore, with the recombinant MVA according to the invention it isnow possible to stably express multiple HIV proteins per recombinant MVAvirus. The expression of multiple proteins, instead of one HIV proteinper virus, is able to induce a wide range of immune responses. Thus, thelikelihood is increased that a protective immune response is generatedthat is effective against different HIV isolates.

To achieve the objects in accordance with the purpose of the invention,as embodied and broadly described herein, the invention comprises thefollowing elements.

A recombinant Modified Vaccinia Ankara (MVA) virus comprising one ormore human immunodeficiency virus DNA coding sequences inserted into oneor more of newly described intergenic regions (IGRs) of the viralgenome.

A recombinant MVA virus wherein the HIV coding sequences are insertedunder the transcriptional control of one or more poxviral transcriptioncontrol elements, such as, for example, the ATI promoter, or the p7.5promoter.

A method for inducing an immune response comprising: (a) providing acomposition comprising a recombinant MVA virus of the invention; and (b)administering the composition to a subject animal, for example, to ahuman.

A method of producing an HIV-1 protein, polypeptide, peptide, antigen,or epitope in vitro comprising the steps of: (a) infection of a hostcell with the recombinant MVA virus of the invention; and (b)cultivation of the infected host cell under suitable conditions toproduce the polypeptide, protein, peptide, antigen, or epitope.

A method of introducing an HIV DNA sequence into a cell ex vivocomprising: (a) infecting the cell with a recombinant MVA virus of theinvention; and, optionally, (b) administering the infected cell to asubject animal, for example, to a human.

A method of introducing an HIV DNA sequence into a subject, said methodcomprising administering a recombinant MVA of the invention to a subjectanimal.

A host cell comprising a recombinant MVA of the invention, wherein thehost cell is chosen from a prokaryotic cell and a eukaryotic cell, suchas, for example, a human cell, a non-human mammalian cell, an insectcell, a fish cell, a plant cell, or a fungal cell.

Additional objects and advantages of the invention will be set forth inpart in the description that follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is more thoroughly understood in view of the drawings, inwhich:

FIGS. 1A-1C illustrate restriction maps of the vector constructs pBNX39(FIG. 1A), pBNX70 (FIG. 1B), and pBN84 (FIG. 1C), comprising about 600bp of MVA sequences flanking the insertion site after the I4L ORF. Theplasmids additionally comprise exogenous DNA (Ecogpt and hBFP) under thetranscriptional control of a poxvirus promoter (P or Ps) between theflanking sequences: Flank 1 (F1 I4L-I5L) and Flank 2 (F2 I4L-I5L). F1rpt stands for a repetitive sequence of Flank 1 to allow deletion of thereporter cassette from a resulting recombinant virus. pBN84 (FIG. 1C)additionally codes for the Dengue virus NS1 protein (NS1 DEN). Furtherabbreviations: AmpR=Ampicilin resistance gene; bps=base pairs;Ecogpt=phosphoribosyltransferase gene isolated from E. coli; andhBFP=sequence for blue fluorescence protein.

FIGS. 2A-2C illustrate restriction maps of the vector constructs pBNX51(FIG. 2A), pBNX67 (FIG. 2B), and pBN27 (FIG. 2C), comprising about 600bp of MVA sequences flanking the insertion site after the ORF 137L(Flank 1:F1 136-137 corresponds to position 129340-129930 of the MVAgenome; Flank 2: F2 136-137 corresponds to position 129931-130540 of theMVA genome).

Additionally, the vector pBNX67 (FIG. 2B) comprises exogenous DNA (NPTII gene=neomycin resistance) under the transcriptional control of apoxvirus promoter (P or Ps) between the flanking sequences. F2rpt standsfor a repetitive sequence of Flank 2 to allow deletion of the reportercassette from a resulting recombinant virus. pBN27 (FIG. 2C)additionally codes for the Dengue virus PrM4 under control of a poxviruspromoter. Further abbreviations: AmpR=Ampicilin resistance gene;bps=base pairs; IRES=internal ribosomal entry site; EGFP=gene for theenhanced green fluorescent protein.

FIGS. 3A-3E illustrate restriction maps of the vector constructs pBNX79(FIG. 3A), pBNX86 (FIG. 3B), pBNX88, (FIG. 3C), pBN34 (FIG. 3D), andpBN56 (FIG. 3D), comprising about 600 bps of MVA sequences flanking theinsertion site between the ORF 007R and 008L (Flank 1: F1 IGR 07-08starts at position 12200 of the MVA genome; Flank 2: F2 IGR 07-08 stopsat position 13400 of the MVA genome). F2rpt stands for a repetitivesequence of Flank 2 to allow deletion of the reporter cassette from aresulting recombinant virus. Additionally, the vectors pBNX88 (FIG. 3C)and pBNX86 (FIG. 3B) comprise exogenous DNA (BFP+gpt and NPT II+EGFP,respectively) under the transcriptional control of a poxvirus promoter(P) between the flanking sequences. F2rpt stands for a repetitivesequence of Flank 2 to allow deletion of the reporter cassette from aresulting recombinant virus. pBN56 (FIG. 3E) additionally codes for theHIV-1 env protein, and pBN34 (FIG. 3D) contains the Dengue virus PrM2coding sequence, env and PrM2 being under control of the poxviruspromoters prATI and p7.5, respectively. Further abbreviations:AmpR=Ampicilin resistance gene; bps=base pairs.

FIGS. 4A-4C illustrate the restriction maps of the vector constructspBNX80 (FIG. 4A), pBNX87 (FIG. 4B), and pBN47 (FIG. 4C) comprising about600/640 bps of MVA sequences flanking the insertion site between the ORF044L and 045L (Flank 1: F1 IGR44-45 starts at position 36730 of the MVAgenome; Flank 2: F2 IGR44-45 stops at position 37970 of the MVA genome).Additionally the vector pBNX87 (FIG. 4B) comprises exogenous DNA (NPT IIgene+EGFP) under the transcriptional control of a poxvirus promoter (P)between the flanking sequences. F2rpt stands for a repetitive sequenceof Flank 2 to allow deletion of the reporter cassette from a resultingrecombinant virus. pBN47 (FIG. 4C) additionally codes for the Denguevirus PrM3 under the control of a poxvirus promoter (pr7.5). Furtherabbreviations: AmpR=Ampicilin resistance gene; bps=base pairs.

FIGS. 5A-5C illustrate the restriction maps of the vector constructspBNX90 (FIG. 5A), pBNX92 (FIG. 5B), and pBN54 (FIG. 5C), comprisingabout 596/604 bps of MVA sequences flanking the insertion site betweenthe ORF 148R and 149L (Flank 1:F1 IGR148-149 starts at position 136900of the MVA genome; Flank 2:F2 IGR148-149 stops at position 138100 of theMVA genome). Additionally the vector pBNX92 (FIG. 5B) comprisesexogenous DNA (Ecogpt+hBFP) under the transcriptional control of apoxvirus promoter (P) between the flanking sequences. pBN54 (FIG. 5C)additionally codes for the Dengue virus PrM1 under the control of thepoxvirus promoter pr7.5. F2rpt stands for a repetitive sequence of Flank2 to allow deletion of the reporter cassette from a resultingrecombinant virus. Further abbreviations: AmpR=Ampicilin resistancegene; bps=base pairs.

FIG. 6 shows a schematic presentation of the intergenic insertion sitesof MVA (GenBank Accession No. U94848).

FIG. 7 illustrates the results of the PCR analysis of IGR I4L-I5L inrecombinant MVA with the Dengue virus NS1 inserted in the IGR I4L-I5L.Lane “BN” shows the PCR product using MVA-BN empty vector. Using the NS1recombinant MVA, a fragment of bigger size is detectable (1, 2, 3, 4:different concentrations of DNA). M=Molecular weight marker, −=waternegative control.

FIG. 8A illustrates the results of the PCR analysis of IGR 136-137 inrecombinant MVA with the Dengue virus PrM4 inserted in the IGR 136-137.Lane “BN” shows the PCR product using MVA-BN empty vector. Using thePrM4 recombinant MVA, a fragment of bigger size is detectable (mBN23,1/10, 1/100: different concentrations of DNA). 1 kb=Molecular weightmarker, H2O=water negative control, pBN27=plasmid positive control.

FIG. 8B shows the multiple step growth curve for MVA-BN empty vector andthe recombinant MVA with PrM4 inserted in IGR 136-137 (MVA-mBN23).

FIG. 9A illustrates the results of the PCR analysis of IGR 07-08 inrecombinant MVA with the Dengue virus PrM2 inserted in the IGR 07-08.Lane 3 shows the PCR product using MVA-BN empty vector. Using the PrM2recombinant MVA, a fragment of bigger size is detectable (lane 2).M=Molecular weight marker, lane 1=water negative control.

FIG. 9B shows the multiple step growth curve for MVA-BN empty vector andthe recombinant MVA with PrM2 inserted in IGR 07-08 (MVA-mBN25).

FIG. 10 illustrates the results of the PCR analysis of IGR 07-08 inrecombinant MVA, with the HIV env inserted in the IGR 07-08. Lane BNshows the PCR product using MVA-BN empty vector. Using the PrM2recombinant MVA, a fragment of bigger size is detectable (lane 1, 2, 3).M=Molecular weight marker, −=water negative control, +=plasmid positivecontrol.

FIG. 11A illustrates the results of the PCR analysis of IGR 44-45 inrecombinant MVA with the Dengue virus PrM3 inserted in the IGR 44-45.Lane BN shows the PCR product using MVA-BN empty vector. Using the PrM3recombinant MVA, a fragment of bigger size is detectable (lane 1-4,different concentrations of DNA). M=Molecular weight marker, −=waternegative control.

FIG. 11B shows the multiple step growth curve for MVA-BN empty vectorand the recombinant MVA with PrM3 inserted in IGR 44-45 (MVA-mBN28).

FIG. 12A illustrates the results of the PCR analysis of IGR 148-149 inrecombinant MVA with the Dengue virus PrM1 inserted in the IGR 148-149.Lane BN shows the PCR product using MVA-BN empty vector. Using the PrM1recombinant MVA, a fragment of bigger size is detectable (lane 1).M=Molecular weight marker, −=water negative control, +=plasmid positivecontrol.

FIG. 12B shows the multiple step growth curve for MVA-BN empty vectorand the recombinant MVA with PrM1 inserted in IGR 44-45 (MVA-mBN33).

FIGS. 13A-13C illustrate the restriction maps of the recombinationvectors pBN108 (td tat-gag/pol) (FIG. 13A), pBN131 (truncated nef) (FIG.13B), and pBN175 (vif-vpr-vpu-rev) (FIG. 13C).

FIG. 13A illustrates the recombination vector pBN108, which was createdby insertion of expression cassettes for transdominant tat and GagPolfusion protein, each under control of the ATI promoter (prATI), in thePacI site of pBNX67 (see FIG. 2B), and expresses the transdominant tatand gag/pol in the IGR 136-137 of the MVA genome. The resulting vector,pBN108, also contains the NPTII gene for selection.

FIG. 13B illustrates the recombination vector pBN131, created forinsertion of the truncated nef (HIV-IBdelnef) in the IGR I4L-I5L of theMVA genome. pBN131 was created by insertion of an expression cassettecomprising a truncated nef under the control of an ATI promoter in thePacI restriction site of the vector pBNX59.

FIG. 13C illustrates the recombination vector pBN17, created forinsertion of the vif-vpr-vpu-rev fusion protein in the IGR 07-08 of theMVA genome. pBN17 was created by insertion of an expression cassettecomprising the vif-vpr-vpu-rev coding sequence under the control of theATI promoter in the NotI restriction site of the vector pBNX86 (see FIG.3B). Additional abbreviations: marker=EGFP.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to various embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

The term “intergenic regions” (IGRs) of the viral genome refers toregions of the MVA viral genome, wherein the regions are, in turn,located between or, in certain embodiments, are flanked by two adjacentopen reading frames (ORFs) of the MVA genome. For example, if the IGRcomprises a sequence that is not part of either ORF but exists betweenthe two adjacent ORFs, then the IGR is said to be “located between” twoadjacent ORFs. On the other hand, there are situations in which there isno additional sequence that exists between the two adjacent ORFs. Inother words, the two adjacent ORFs abut. In the latter situations then,the IGR does not have a corresponding sequence in itself; rather, theIGR refers to the site, or genomic position, whereby an heterologoussequence can be inserted between the two ORFs, thus enlarging thedistance separating the otherwise abutting ORFs. In this case, the IGRis said to be “flanked by” two adjacent ORFs.

“ORFs of the MVA genome” occur in two coding directions: forward andreverse (for detailed description, see for example, Antoine et al.,Virology 244, 365-396 [1998]), incorporated herein by reference).Consequently, the polymerase activity occurs from left to right, i.e.,forward direction and, correspondingly, from right to left, reversedirection. In certain embodiments of the invention, ORFs occurring inthe forward coding direction are referred to as 5′ORF3′, whereas ORFsoccurring in the reverse coding direction are referred to as 3′FRO5′ tofacilitate the understanding of their orientation in the MVA genome.

It is common practice in poxvirology, and it became a standardclassification for Vaccinia viruses, to identify ORFs by theirorientation and their position on the different HindIII restrictiondigest fragments of the genome (see for example, Goebel et al., Virology179, 247-266 and 517-563, [1990]; and Massung, R. F. et al., Virology201, 215-240 [1994], incorporated herein by reference). For the commonpractice nomenclature, the different HindIII fragments are named bydescending capital letters corresponding with their descending size. TheORFs are numbered from left to right on each HindIII fragment and theorientation of the ORF is indicated by a capital L (standing fortranscription from right to Left) or R (standing for transcription fromleft to Right).

Additionally, there is a more recent publication of the MVA genomestructure, which uses a different nomenclature, simply numbering the ORFfrom the left to the right end of the genome, and indicating theirorientation with a capital L or R (Antoine et al., Virology 244, 365-396[1998]). As an example the I4L ORF, according to the old nomenclature,corresponds to the 064L ORF according to Antoine et al. If not indicateddifferently, the invention uses the nomenclature according to Antoine etal. Accordingly, herein, the IGRs are referred to in one of two ways,depending on the nomenclature used to name the ORFs. For example, an IGRlocated between the two adjacent ORFs, ORF 001 L and ORF 002L, is saidto be IGR 001 L-002L. An IGR located between the two adjacent ORFs, ORFI4L and ORF I5L, is said to be IGR I4L-I5L.

Herein, and according to the old nomenclature, ORF 006L corresponds toC10L, 019L corresponds to C6L, 020L to N1L, 021L to N2L, 023L to K2L,028R to K7R, 029L to F1L, 037L to F8L, 045L to F15L, 050L to E3L, 052Rto ESR, 054R to E7R, 055R to EBR, 056L to E9L, 062L to I1L, 064L to I4L,065L to I5L, 081R to L2R, 082L to L3L, 086R to J2R, 088R to J4R, 089L toJSL, 092R to H2R, 095R to HSR, 107R to D10R, 108L to D11L, 122R to A11R,123L to A12L, 125L to A14L, 126L to A15L, 135R to A24R, 136L to A25L,137L to A26L, 141L to A30L, 148R to A37R, 149L to A38L, 152R to A40R,153L to A41L, 154R to A42R, 157L to A44L, 159R to A46R, 160L to A47L,165R to A56R, 166R to A57R, 167R to B1R, 170R to B3R, 176R to B8R, 180Rto B12R, 184R to B16R, 185L to B17L, and 187R to B19R.

Accordingly, IGR I4L-I5L (old nomenclature) corresponds to IGR 064L-065L(new nomenclature), and refers to the intergenic region located between,or flanked by, ORFs I4L and I5L.

Furthermore, unless immediately preceeded by the term “IGR” and thusspecified as referring to an IGR, the use of the term I4L-I5L refers tothe pair of ORFs I4L and I5L; it is not to be confused with IGR I4L-I5L,which refers to the region located in between, or flanked by, the ORFsI4L and I5L. By analogy, the use of the expression, for example, “agroup of ORFs selected from 001 L-002L, 002L-003L, 005R-006R,” issynonymous with the use of the expression, and refers to, “a group ofpairs of ORFs selected from the pair 001 L and 002L; the pair 002L and003L; and the pair 005R and 006R.”

In one embodiment, reference is made to the various HIV sequences asdisclosed in the GenBank database, in particular to the sequence of theHIV-1 isolate HXB2R having the GenBank accession number K03455.

The term “derivative of the amino acid sequence of an HIV protein,” asused in the present specification, refers to HIV proteins that have analtered amino acid sequence compared to the corresponding naturallyoccurring HIV protein. An altered amino acid sequence may be a sequencein which one or more amino acids of the sequence of the HIV protein aresubstituted, inserted, or deleted; for example, the derivative can haveone or more conservative amino acid substitutions. More particularly, a“derivative of the amino acid sequence of an HIV protein” is an aminoacid sequence showing an identity of at least 50%, such as of at least70%, of at least 80%, or even of at least 90%, when the correspondingpart of the amino acid sequence in the fusion protein is compared to theamino acid sequence of the respective HIV protein of known HIV isolates.

According to the invention, an amino acid sequence is regarded as havingthe above indicated sequence identity even if the identity is found forthe corresponding protein of only one HIV isolate, irrespective of thefact that there might be corresponding proteins in other isolatesshowing a lower identity. By way of example, if a Vpr derivative in thefusion protein shows an identity of 95% to the Vpr sequence of one HIVisolate, but only an identity of 50-70% to (all) other HIV isolates, theidentity of said Vpr derivative is regarded as being of at least 90%.

In a preferred embodiment, the term “derivative of an HIV protein”refers to an amino acid sequence showing an identity of at least 50%,70%, 80%, or 90% to the respective HIV protein in the HIV-1 isolateHXB2R (GenBank accession number K03455).

The percent identity may be determined, for example, by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Other programs used by one skilled in the art of sequencecomparison may also be used.

New Sites for Insertion of Exogenous DNA Sequences into the MVA Genome

In one embodiment, the invention encompasses new sites for the insertionof exogenous DNA sequences into the genome of Modified Vaccinia Ankara(MVA) virus. The new insertion sites are located in the intergenicregions (IGRs) of the viral genome, wherein the IGRs are, in turn,either located between, or are flanked by, two adjacent open readingframes (ORFs) of the MVA genome.

Accordingly, in certain embodiments, the invention relates torecombinant MVA viruses comprising one or more HIV DNA sequencesinserted into one or more of the IGRs of the invention.

It was surprisingly found that exogenous DNA sequences remain stablewhen inserted into IGRs of the MVA genome. These results were unexpectedbecause, as already indicated above, the genome of MVA reportedly isunstable. Reportedly, genes or DNA sequences non-essential forpropagation of the virus are deleted or fragmented. Indeed, whereas ithas also been reported that stable recombinant MVAs are obtained whenheterologous DNA sequences are inserted into the naturally occurringdeletion sites of the MVA genome (PCT/EP96/02926), which was anothersurprising observation in itself. Typically, host range genes are notsuitable insertion sites in MVAs. Such is the case of, for example, thetk-locus widely used for the generation of other recombinant poxviruses.The fact that Vero-MVA has one extra genomic deletion (PCT/EP01/02703)also suggests that the genome is dynamic, in the sense that it readilydeletes genes that are not required for propagation. Therefore, andcontrary to the results of the invention, one skilled in the art wouldhave reasonably expected that heterologous DNA sequences non-essentialfor viral propagation, when inserted into spaces between ORFs, would bedeleted by the MVA virus as well.

While the nucleotide sequence of an ORF encodes an amino acid sequenceforming a peptide, polypeptide, or protein, the IGRs between two ORFshave no coding capacity. Accordingly, in certain embodiments, the IGRsmay comprise regulatory elements, binding sites, promoter and/orenhancer sequences essential for, or involved in, the transcriptionalcontrol of the viral gene expression. Thus, the IGR may be involved inthe regulatory control of the viral life cycle.

In further embodiments, however, the inventors of the invention havealso found that the newly identified insertion sites have the unexpectedadvantage that exogenous DNA sequences can be stably inserted into theMVA genome, and furthermore, have such capability without influencing orchanging the typical characteristics and gene expression of MVA (forexample, see FIGS. 6 through 12). The new insertion sites also areespecially useful because no ORF or coding sequence of MVA is altered bythe process of insertion.

Moreover, it was surprisingly found that the expression level of aforeign gene inserted into an IGR is higher than the expression level ofa foreign gene inserted into a deletion site of the MVA genome (see alsoExample 1).

According to the invention, the nucleotide sequence of an ORF shouldstart with a start codon and end with a stop codon. Depending on theorientation of the two adjacent ORFS, the IGR, i.e., the region inbetween these ORFS, is flanked by one of the following: the two stopcodons of the two adjacent ORFS; the two start codons of the twoadjacent ORFS; the stop codon of the first ORF and the start codon ofthe second ORF; or the start codon of the first ORF and the stop codonof the second ORF.

Accordingly, in one embodiment, the site for insertion of the exogenousDNA sequence into the IGR is downstream, i.e. 3′, of the stop codon of afirst ORF; and, in case the adjacent ORF, also termed second ORF, hasthe same orientation as the first ORF, the insertion site further liesupstream, i.e. 5′, of the start codon of the second ORF. Thisarrangement can be represented as: 5′ORF3′-IGR-5′ORF3′.

In a further embodiment, the site for insertion of the exogenous DNAsequence into the IGR is downstream, i.e. 3′, of the stop codon of afirst ORF; and, in case the second ORF has an opposite orientationrelative to the first ORF, which means the orientation of the twoadjacent ORFs points to each other, the insertion site further liesdownstream of the stop codons of both ORFs. This arrangement can berepresented as: 5′ORF3′-IGR-3′FRO5′.

In yet a further embodiment, in case the two adjacent ORFs read in thesame direction from right to left of the viral genome, which issynonymous with a positioning that is characterized in that the startcodon of the first ORF is adjacent to the stop codon of the second ORF,then the exogenous DNA is inserted upstream (or 5′) of one start codonand downstream (or 3′) from the other. This arrangement can berepresented as: 3′FRO5′-IGR-3′FRO5′.

In yet a further embodiment, in case the two adjacent ORFs read inopposite direction, but the orientation of the two adjacent ORFs pointsaway from each other, which is synonymous with a positioning that ischaracterized in that the start codons of the two ORFs are adjacent toeach other, then the exogenous DNA is inserted upstream relative to bothstart codons. This arrangement can be represented as:3′FRO5′-IGR-5′ORF3′.

In one embodiment, heterologous DNA sequences can be inserted into oneor more IGRs in between two adjacent ORFs, said IGR selected from thegroup comprising:

001 L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L,017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L,025L-026L, 028R-029L, 030L-031 L, 031 L-032L, 032L-033L, 035L-036L,036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R,049L-050L, 050L-051 L, 051 L-052R, 052R-053R, 053R-054R, 054R-055R,055R-056L, 061 L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R,078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R,088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R,101R-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L,110L-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R,122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R,136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R,146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 154R-155R,156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R,164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R,175R-176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R,184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R,192R-193R.

In a preferred embodiment, the heterologous sequence is inserted into anIGR flanked by two adjacent ORFs selected from the group comprising007R-008L, 018L-019L, 044L-045L, 064L-065L, 136L-137L, 148R-149L.

Recombinant MVA Comprising DNA Sequences Inserted into Novel IGRs MVAViruses

In a preferred embodiment, the recombinant MVA virus of the invention isreplication incompetent in humans and non-human primates. The terms MVAvirus that is “replication incompetent” in humans and/or non-humanprimates, and the synonymous term virus that is “not capable of beingreplicated to infectious progeny virus” in humans and/or non-humanprimates, both refer preferably to MVA viruses that do not replicate atall in the cells of the human and/or non-human primate vaccinated withsaid virus. However, also within the scope of the present applicationare those viruses that show a minor residual replication activity thatis controlled by the immune system of the human and/or non-human primateto which the recombinant MVA virus is administered.

In one embodiment, the replication incompetent recombinant MVA virusesmay be viruses that are capable of infecting cells of the human and/ornon-human primate in which the virus is used as vaccine. Viruses thatare “capable of infecting cells” are viruses that are capable ofinteracting with the host cells to such an extent that the virus, or atleast the viral genome, becomes incorporated into the host cell.Although the viruses used according to the invention are capable ofinfecting cells of the vaccinated human and/or non human primate, theyare not capable of being replicated to infectious progeny virus in thecells of the vaccinated human and/or non-human primate.

According to the invention, it is to be understood, that a virus that iscapable of infecting cells of a first animal species, but is not capableof being replicated to infectious progeny virus in said cells, maybehave differently in a second animal species. For example, MVA-BN andits derivatives (see below) are viruses that are capable of infectingcells of the human, but that are not capable of being replicated toinfectious progeny virus in human cells. However, the same viruses areefficiently replicated in chickens; i.e., in chickens, MVA-BN is a virusthat is both capable of infecting cells and capable of being replicatedto infectious progeny virus in those cells.

A suitable test that allows one to predict whether a virus is capable ornot capable of being replicated in humans is disclosed in WO 02/42480(incorporated herein by reference) and uses the severely immunecompromised AGR129 mice strain. Furthermore, instead of the AGR129 mice,any other mouse strain can be used that is incapable of producing matureB and T cells, and as such is severely immune compromised and highlysusceptible to a replicating virus. The results obtained in this mousemodel reportedly are indicative for humans and, thus, according to thepresent application, a virus that is replication incompetent in saidmouse model is regarded as a virus that is “replication incompetent inhumans.”

In other embodiments, the viruses according to the invention arepreferably capable of being replicated in at least one type of cells ofat least one animal species. Thus, it is possible to amplify the virusprior to its administration to the animal that is to be vaccinatedand/or treated. By way of example, reference is made to MVA-BN that canbe amplified in CEF (chicken embryo fibroblasts) cells, but that is avirus that is not capable of being replicated to infectious progenyvirus in humans.

In further embodiments, Modified Vaccinia virus Ankara (MVA) is suitablefor use in humans and several animal species such as mice and non-humanprimates. MVA is known to be exceptionally safe. MVA has been generatedby long-term serial passages of the Ankara strain of Vaccinia virus(CVA) on chicken embryo fibroblasts (for review see Mayr, A.,Hochstein-Mintzel, V. and Stickl, H. Infection 3, 6-14 [1975]; SwissPatent No. 568,392). Examples of MVA virus strains that have beendeposited in compliance with the requirements of the Budapest Treaty,and that are useful for the generation of recombinant viruses accordingto the invention, are strains MVA 572 deposited at the EuropeanCollection of Animal Cell Cultures (ECACC), Salisbury (UK) with thedeposition number ECACC 94012707 on Jan. 27, 1994; MVA 575 depositedunder ECACC 00120707 on Dec. 7, 2000; and MVA-BN deposited with thenumber 00083008 at the ECACC on Aug. 30, 2000.

In one embodiment, the MVA strain used in generating a recombinant MVAis MVA-575, or a derivative thereof.

In a preferred embodiment, the MVA strain used in generating arecombinant MVA is MVA-572, or a derivative thereof.

In another preferred embodiment, the MVA strain used in generating arecombinant MVA is MVA-Vero, or a derivative thereof. MVA-Vero strainshave been deposited at the European Collection of Animal Cell Culturesunder the deposition numbers ECACC V99101431 and 01021411. The safety ofthe MVA-Vero is reflected by its biological, chemical and physicalcharacteristics as described in the International Patent ApplicationPCT/EP01/02703, incorporated herein by reference in its entirety. Incomparison to other MVA strains, the Vero-MVA includes one additionalgenomic deletion.

In a more preferred embodiment, the MVA strain is MVA-BN, or aderivative thereof. MVA-BN has been deposited at the European Collectionof Animal Cell Cultures with the deposition number ECACC V00083008.MVA-BN virus is an extremely attenuated virus also derived from ModifiedVaccinia Ankara virus. A definition of MVA-BN and its derivatives isgiven in PCT/EP01/13628, incorporated herein by reference in itsentirety.

The term “derivatives” of a virus according to the invention refers toprogeny viruses showing the same characteristic features as the parentvirus, but showing differences in one or more parts of its genome. Theterm “derivative of MVA” describes a virus which has the same functionalcharacteristics compared to MVA. For example, a derivative of MVA-BN hasthe characteristic features of MVA-BN. One of these characteristics ofMVA-BN, or of derivatives thereof, is its attenuation and lack ofreplication in human cell lines, such as the human keratinocyte cellline HaCaT, the human embryo kidney cell line 293, the human boneosteosarcoma cell line 143 B, and the human cervix adenocarcinoma cellline HeLa.

In a preferred embodiment, the virus according to the invention is avirus that has been produced and/or passaged under serum free conditionsto reduce the risk of infections with agents contained in serum. Oneskilled in the art of the invention is familiar with methods forproducing and/or passaging virus under serum free conditions.

In certain embodiments, recombinant MVA, or a derivative thereof,according to the invention, is administered in a concentration range ofabout 10⁴ to about 10⁹ TCID₅₀/ml, preferably in a concentration range ofabout 10⁵ to about 5×10⁸ TCID₅₀/ml, most preferably in a concentrationrange of about 10⁶ to about 10⁸ TCID₅₀/ml.

The actual amount used depends on the type of virus and the animalspecies to be vaccinated. For MVA-BN, a typical vaccination dose forhumans comprises about 5×10⁷ TCID₅₀ to about 5×10⁸ TCID₅₀, such as about1×10⁸ TCID₅₀, administered subcutaneously.

In one embodiment, an immune response is induced with a singleadministration of the recombinant poxvirus as defined above, inparticular with an MVA strain, such as MVA-BN and its derivatives.Accordingly, one may use the MVA virus according to the invention, inparticular an MVA strain, such as MVA-BN and its derivatives inhomologous prime boost regimes. In these regimes, it is possible to usea recombinant poxvirus such as a recombinant MVA for a firstvaccination, and to boost the immune response generated in the firstvaccination by further administration of the same virus, or of a relatedrecombinant MVA virus, than the one used in the first vaccination.

In another embodiment, the recombinant poxvirus according to theinvention, in particular an MVA strain, such as MVA-BN and itsderivatives, may also be used in heterologous prime-boost regimes; theseregimes are those in which one or more of the vaccinations is done witha MVA virus as defined above, and one or more of the additionalvaccinations is done with another type of vaccine, for example, anothervirus vaccine, a protein or a nucleic acid vaccine.

According to the invention, the mode of administration may beintravenously, intradermal, intranasal, or subcutaneously. A preferredembodiment is subcutaneous administration. However, any other mode ofadministration may be used such as, for example, scarification.

In one embodiment, the recombinant MVA according to the invention isuseful as a medicament or vaccine.

According to a preferred embodiment, the recombinant MVA is used for theintroduction of an exogenous coding sequence into a target cell, saidsequence being either homologous or heterologous to the genome of thetarget cell.

In one embodiment, the introduction of an exogenous coding sequence intoa target cell is done in vitro to produce proteins, polypeptides,peptides, antigens or antigenic epitopes.

In a preferred embodiment, the method of introduction of an exogenouscoding sequence into a target cell in vitro to produce proteins,polypeptides, peptides, antigens or antigenic epitopes comprises theinfection of a host cell with the recombinant MVA according to theinvention; cultivation of the infected host cell under suitableconditions; and isolation and/or enrichment of the polypeptide, peptide,protein, antigen, epitope and/or virus produced by said host cell.

In a further embodiment, the method for introduction of exogenoussequences into cells is applied for in vitro and/or in vivo therapy.

In one embodiment, for in vitro therapy, isolated cells that have beenpreviously (ex vivo) infected with the recombinant MVA according to theinvention are administered to the living animal body for affecting,preferably for inducing, an immune response.

In another embodiment, for in vivo therapy, the recombinant MVA virusaccording to the invention is directly administered to the living animalbody for affecting, preferably for inducing, an immune response.

In a preferred embodiment, the cells surrounding the site ofinoculation, and also cells where the virus is transported to via, forexample, the blood stream, are directly infected in vivo by therecombinant MVA according to the invention. After infection, these cellssynthesize the proteins, peptides or antigenic epitopes of thetherapeutic genes, which are encoded by the exogenous coding sequences,and subsequently, present them or parts thereof on the cellular surface.Subsequently, specialized cells of the immune system recognize thepresentation of such heterologous proteins, peptides or epitopes andlaunch a specific immune response.

Since the MVA is highly growth-restricted and, thus, highly attenuated,it is useful for the treatment of a wide range of mammals includinghumans, and particularly immune-compromised animals or humans. Thus, inone embodiment, the invention also provides pharmaceutical compositionsand vaccines for inducing an immune response in a living animal body,including a human.

In one embodiment, the pharmaceutical composition may generally includeone or more pharmaceutical acceptable and/or approved carriers,additives, antibiotics, preservatives, adjuvants, diluents and/orstabilizers. Non-limiting examples of such auxiliary substances arewater, saline, glycerol, ethanol, wetting or emulsifying agents, pHbuffering substances, or the like. Suitable carriers are typicallyselected from the group comprising large, slowly metabolized moleculessuch as, for example, proteins, polysaccharides, polylactic acids,polyglycolitic acids, polymeric amino acids, amino acid copolymers,lipid aggregates, or the like.

In another embodiment, for the preparation of vaccines, the recombinantMVA virus according to the invention is converted into a physiologicallyacceptable form. This can be done based on the experience in thepreparation of poxvirus vaccines used for vaccination against smallpox(as described by Stickl, H. et al. Dtsch. med. Wschr. 99, 2386-2392[1974]). For example, the purified virus is stored at −80° C. with atiter of 5×10⁸ TCID₅₀/ml formulated in 10 mM Tris, 140 mM NaCl pH 7.4.

In one embodiment, the MVA virus according to the invention is used forthe preparation of vaccine shots. For example, about 10² to about 10⁸particles of the virus are lyophilized in 100 ml of phosphate-bufferedsaline (PBS) in the presence of 2% peptone and 1% human albumin in anampoule, preferably a glass ampoule. In another non-limiting example,the vaccine shots are produced by stepwise freeze-drying of the virus ina formulation. In certain embodiments, this formulation can containadditional additives such as mannitol, dextran, sugar, glycine, lactoseor polyvinylpyrrolidone or other aids, such as antioxidants or inertgas, stabilizers or recombinant proteins (for example, human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no immediate need exists, the ampoule isstored preferably at temperatures below −20° C.

In a further embodiment, for vaccination or therapy, the lyophilisatecan be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferablyphysiological saline or Tris buffer, and administered eithersystemically or locally, i.e. parenterally, subcutaneously,intramuscularly, by scarification or any other path of administrationknow to the skilled practitioner. The mode of administration, the doseand the number of administrations can be optimized by those skilled inthe art in a known manner. However, most commonly, a patient isvaccinated with a second shot about one month to six weeks after thefirst vaccination shot.

Sequences Derived from the MVA Genome

The invention further relates to plasmid vectors, which can be used togenerate recombinant MVA according to the invention, and also relates tocertain DNA sequences.

In one embodiment, the IGR located between two adjacent ORFs comprisesnucleotide sequences, herein referred to as “IGR-DNA sequences”, inwhich the exogenous DNA sequence of interest can be inserted.

Accordingly, in one embodiment, the plasmid vector according to theinvention comprises a DNA sequence derived from, or homologous to, thegenome of MVA, wherein said DNA sequence comprises a complete or partialfragment of an IGR-DNA sequence.

In a preferred embodiment, the plasmid vector further comprises,inserted into said IGR-derived sequence, at least one cloning site for(i) the insertion of an exogenous DNA sequence of interest and,preferably, for (ii) the insertion of a poxviral transcription controlelement operatively linked to said exogenous DNA sequence. Optionally,the plasmid vector further comprises a reporter- and/orselection-gene-cassette.

In a preferred embodiment, the plasmid vector further comprisessequences of the two adjacent ORFs flanking said complete or partialfragment of the IGR-DNA sequence.

In another embodiment, some IGRs have been identified, which do notinclude nucleotide sequences. As described above (see Definitions),these IGRs are insertion sites flanked by abuting ORFs. Thus, in thisembodiment, the plasmid vector comprises DNA sequences of the “IGRflanking sequences”, i.e., DNA sequences of the two adjacent ORFs.

In a preferred embodiment, the cloning site for the insertion of theexogenous DNA sequence is inserted into the IGR.

In some embodiments, the DNA of the IGR flanking sequences is used todirect the insertion of exogenous DNA sequences into the correspondingIGR in the MVA genome.

In a more preferred embodiment, such a plasmid vector may additionallyinclude a complete or partial fragment of an IGR sequence whichcomprises the cloning site for the insertion of the heterologous DNAsequence and, optionally, of the reporter- and/orselection-gene-cassette.

In one embodiment, IGR-DNA sequences are preferably selected from IGRsselected from the group comprising:

001 L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L,017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L,025L-026L, 028R-029L, 030L-031 L, 031 L-032L, 032L-033L, 035L-036L,036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R,049L-050L, 050L-051 L, 051 L-052R, 052R-053R, 053R-054R, 054R-055R,055R-056L, 061 L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R,078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R,088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R,101R-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L,110L-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R,122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R,136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R,146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 154R-155R,156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R,164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R,175R-176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R,184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R,192R-193R.

In another embodiment, IGR flanking sequences of the two adjacent ORFsare preferably selected from ORFs selected from the group comprising:

001 L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L,017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L,025L-026L, 028R-029L, 030L-031 L, 031 L-032L, 032L-033L, 035L-036L,036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R,049L-050L, 050L-051 L, 051 L-052R, 052R-053R, 053R-054R, 054R-055R,055R-056L, 061 L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R,078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R,088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R,101R-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L,110L-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R,122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R,136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R,146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 154R-155R,156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R,164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R,175R-176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R,184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R,192R-193R.

In a preferred embodiment, IGR-DNA sequences, as well as IGR flankingsequences, are selected from IGRs and ORFs, respectively, selected fromthe group comprising 007R-008L, 018L-019L, 044L-045L, 064L-065L,136L-137L, 148L-149L.

In another preferred embodiment, IGR-DNA sequences are selected from thegroup comprising the nucleotide sequences:

no. 527-608 of SEQ ID NO: 32;

no. 299-883 of SEQ ID NO: 33;

no. 339-852 of SEQ ID NO: 34;

no. 376-647 of SEQ ID NO: 35;

no. 597-855 of SEQ ID NO: 36;

no. 400-607 of SEQ ID NO: 37.

In another preferred embodiment, IGR flanking sequences are selectedfrom the group comprising the nucleotide sequences:

no. 1-525 and 609-1190 of SEQ ID NO: 32;

no. 101-298 and 884-1198 of SEQ ID NO: 33;

no. 1-338 and 853-1200 of SEQ ID NO: 34;

no. 1-375 and 648-1200 of SEQ ID NO: 35;

no. 1-596 and 856-1200 of SEQ ID NO: 36;

no. 1-399 and 608-1081 of SEQ ID NO: 37.

In yet another preferred embodiment, the DNA sequences are preferablyderived from, or are homologous to, sequences of the genome of the MVAas deposited at ECACC under deposition number V00083008.

In yet another embodiment, the DNA sequences according to the inventionare used to (i) identify or isolate the MVA or its derivatives accordingto the invention, and/or (ii) identify cells or individuals infectedwith an MVA according to the invention.

In another embodiment, the DNA sequences according to the invention areused to generate PCR-primers and/or hybridization probes.

In yet another embodiment, the DNA sequences according to the inventionare used in array technologies.

Generation of Recombinant MVA

Methods suitable to generate a plasmid vector according to the inventionare familiar to those skilled in the art of the invention. For example,to generate a plasmid vector with the sequences of the invention, thesequences can be isolated and cloned into a standard cloning vector,such as pBluescript (Stratagene), wherein they flank the exogenous DNAto be inserted into the MVA genome. Optionally, such a plasmid vectorcomprises a selection- or reporter-gene cassette, which can be deletedfrom the final recombinant virus, due to a repetitive sequence includedinto said cassette.

Methods to introduce exogenous DNA sequences by a plasmid vector into anMVA genome and methods to obtain recombinant MVA are well known to theperson skilled in the art and, additionally, can be deduced from thefollowing references:

-   -   (i) Molecular Cloning, A Laboratory Manual, Second Edition,        by J. Sambrook, E. F. Fritsch and T. Maniatis. Cold Spring        Harbor Laboratory Press, 1989: describes techniques and know how        for standard molecular biology techniques such cloning of DNA,        RNA isolation, western blot analysis, RT-PCR and PCR        amplification techniques;    -   (ii) Virology Methods Manual, edited by Brian W. J. Mahy and        Hillar O. Kangro, Academic Press, 1996: describes techniques for        the handling and manipulation of viruses;    -   (iii) Molecular Virology: A Practical Approach, edited by A J        Davison and R M Elliott, The Practical Approach Series, IRL        Press at Oxford University Press, Oxford 199, Chapter 9,        Expression of genes by Vaccinia virus vectors; and    -   (iv) Current Protocols in Molecular Biology, Publisher: John        Wiley and Son Inc., 1998, Chapter 16, section IV: Expression of        proteins in mammalian cells using Vaccinia viral vector:        describes techniques and know-how for the handling, manipulation        and genetic engineering of MVA.

In one embodiment, the MVA and derivatives thereof, according to theinvention, preferably the MVA deposited at ECACC under deposition numberV00083008, is produced by transfecting a cell with a plasmid vectoraccording to the invention, infecting the transfected cell with an MVAand, subsequently, identifying, isolating and, optionally, purifying theMVA according to the invention.

Exogenous Sequences For Integration into Novel IGRs

Heterologous or exogenous DNA sequences are terms that are usedinterchangeably herein and refer to sequences which, in nature, are notnormally found associated with the poxvirus as used according to theinvention.

Thus, according to a further embodiment of the invention, the exogenousDNA sequence comprises at least one coding sequence. The coding sequenceis operatively linked to a transcription control element, preferably toa poxviral transcription control element. In another embodiment, furthercombinations between poxviral transcription control element and, forexample, internal ribosomal entry sites are used (for exemplary details,see FIGS. 1-13 and Examples 1-6).

According to another embodiment, the exogenous DNA sequence can comprisetwo or more coding sequences linked to one or several transcriptioncontrol elements. Preferably, the coding sequence encodes one or moreproteins selected from the group comprising polypeptides, peptides,foreign antigens or antigenic epitopes, especially those oftherapeutically interesting genes.

“Therapeutically interesting genes” according to the invention can begenes derived from, or homologous to, genes of pathogenous or infectiousmicroorganisms, which are disease-causing. Accordingly, in the contextof the invention, such therapeutically interesting genes are presentedto the immune system of an organism in order to affect, or morepreferably to induce, a specific immune response and, thereby, vaccinateor prophylactically protect the organism against an infection with themicroorganism.

In one embodiment, therapeutically interesting genes according to theinvention comprise disease related genes, which have a therapeuticeffect on proliferative disorders, cancer, or metabolic diseases. Forexample, a therapeutically interesting gene regarding cancer could be acancer antigen that has the capacity to induce a specific anti-cancerimmune reaction.

In a further preferred embodiment of the invention, the therapeuticallyinteresting genes are selected from genes of infectious viruses, suchas, for example, Dengue virus, Japanese encephalitis virus, Hepatitisvirus B or C, or immunodeficiency viruses, such as HIV.

In one embodiment, genes derived from Dengue virus are preferably NS1and PrM genes, wherein the genes can be derived from one, two, three orfrom all of the four reported Dengue virus serotypes. The NS1 gene ispreferably derived from Dengue virus serotype 2 and is preferablyinserted into the IGR between the ORFs 064L-065L (I4L-I5L). PrM genes,preferably derived from all of the four Dengue virus serotypes, arepreferably inserted into the IGRs between the ORFs selected from007R-008L, 044L-045L, 136L-137L, 148R-149L. More preferably, the PrMgene derived from Dengue virus serotype 1 (prM 1) is inserted into IGR148R-149L, PrM 2 into IGR 007R-008L, PrM 3 into IGR 044L-045L, and PrM 4into IGR 136L-137L.

According to a further embodiment of the invention, the exogenous DNAsequence comprises a coding sequence, which comprises at least onemarker or selection gene.

“Marker gene” or genes, induce a color reaction in transduced cells,which can be used to identify transduced cells. The skilled practitioneris familiar with a variety of marker genes, which can be used in apoxviral system. Among these are the genes encoding, for example,β-Galactosidase (β-3-gal), β-3-Glucosidase (β-glu), Enhanced GreenFluorescence protein (EGFP), or Blue Fluorescence Protein (BFP or hbfp).

“Selection gene” or genes, transduce a particular resistance to a cell,whereby a certain method for selecting such cell becomes possible. Theskilled practitioner is familiar with a variety of selection genes,which can be used in a poxviral system. Among these are, for example,Neomycin resistance gene (NPT) or Phosphoribosyl transferase gene (gpt).

According to a further embodiment of the invention, the exogenous DNAsequence comprises a spacer or spacing sequence, which separatespoxviral transcription control element and/or coding sequence in theexogenous DNA sequence from the stop codon and/or the start codon of theadjacent ORFs.

According to the invention, this spacer or spacing sequence, locatedbetween the stop/start codon of the adjacent ORF and the coding sequenceinserted in the exogenous DNA, has the advantage of stabilizing theinserted exogenous DNA and, thus, any resulting recombinant virus. Thesize of a suitable spacer sequence is variable, as long as the sequenceis without its own coding or regulatory function.

According to a further embodiment, the spacer sequence, separating thepoxviral transcription control element and/or the coding sequence in theexogenous DNA sequence from the stop codon of the adjacent ORF, is atleast one nucleotide long.

According to yet another embodiment, the spacing sequence, separatingthe poxviral transcription control element and/or the coding sequence inthe exogenous DNA sequence from the start codon of the adjacent ORF, isat least 30 nucleotides.

In one embodiment, if a typical Vaccinia virus promoter element isupstream of a start codon, the insertion of exogenous DNA might separatethe promoter element from the start codon of the adjacent ORF. A spacingsequence of about 30 nucleotides is the preferred distance to securethat, a poxviral promoter located upstream of the start codon of theORF, is not influenced.

Additionally, according to a further preferred embodiment, the distancebetween the inserted exogenous DNA and the start codon of the adjacentORF comprises about 50 nucleotides, and more preferably about 100nucleotides.

“A typical Vaccinia promoter” element can be identified by scanning for,for example, the sequence “TAAAT” for late promoters (Davison & Moss, J.Mol. Biol. 210: 771-784 [1989]), and for an NT rich domain for earlypromoters.

According to a further preferred embodiment, the spacing sequencecomprises an additional poxviral transcription control element, which iscapable of controlling the transcription of the adjacent ORF.

Recombinant MVA Viruses Expressing HIV Peptides Inserted into IGRs

In a preferred embodiment, the invention relates to recombinant MVAviruses comprising one or a plurality of exogenous sequences in theviral genome, selected from a group consisting of expression cassettescomprising one or more HIV proteins; expression cassettes comprising oneor more parts of HIV proteins; and expression cassettes comprising oneor more derivatives of HIV proteins.

According to one embodiment, the exogenous sequences comprise HIVpeptides selected from a group consisting of the full-length proteinsGag (capsid protein), Pol (polymerase protein), Env (envelope protein),Tat, Vif, Vpu, Vpr, Rev and Nef; and parts or derivatives thereof.

For example, according to one embodiment, the exogenous DNA sequence isderived from HIV and encodes HIV env, wherein the HIV env gene ispreferably inserted into the IGR 007R-008L.

In certain embodiments, the recombinant MVA virus, in particular MVA-BNand its derivatives, comprises regulatory/accessory proteins of HIV. Theprotein can exhibit full biological activity.

The regulatory/accessory proteins of HIV proteins have a biologicalactivity that can have undesired side effects. Thus, it is also withinthe scope of the invention that, in certain other embodiments, one ormore HIV proteins expressed from the recombinant MVA virus has a reducedbiological activity compared to the wild-type protein, and thus is saidto have reduced activity.

One skilled in the art is familiar with tests suitable to determinewhether an HIV protein has reduced activity.

The molecular mechanism of the Vif protein, which is essential for viralreplication in vivo, remains unknown, but Vif possesses a strongtendency toward self association. This multimerization was shown to beimportant for Vif function in viral life cycle (Yang S. et al., J BiolChem 276: 4889-4893 [2001]). Additionally, vif was shown to bespecifically associated with the viral nucleoprotein complex, and thismight be functionally significant (Khan M. A. et al., J. Virol. 75 (16):7252-65 [2001]).

Thus, in a preferred embodiment, the exogenous sequence expresses a vifwith reduced activity, and the vif shows a reduced multimerizationand/or association to the nucleoprotein complex.

The Vpr protein plays an important role in the viral life cycle. Vprregulates the nuclear import of the viral pre-integration complex andfacilitates infection of non dividing cells such as macrophages(Agostini et al., AIDS Res Hum Retroviruses 18(4):283-8 [2002]).Additionally, it has transactivating activity mediated by interactionwith the LTR (Vanitharani R. et al., Virology 289 (2):334-42 [2001]).

Thus, in a preferred embodiment, the exogenous sequence expresses a vprwith reduced activity, and said vpr shows either decreased or notransactivation and/or interaction, with the viral preintegrationcomplex.

Vpx, which is highly homologous to Vpr, is also critical for efficientviral replication in non-dividing cells. Vpx is packaged in virusparticles via an interaction with the p6 domain of the gag precursorpolyprotein. Like Vpr, Vpx is involved in the transportation of thepreintegration complex into the nucleus (Mahalingam et al., J. Virol. 75(1):362-74 [2001]).

Thus, in a preferred embodiment, the exogenous sequence expresses a Vpxwith reduced activity, and the Vpx has a decreased ability to associateto the preintegration complex via the gag precursor.

The Vpu protein is known to interact with the cytoplasmic tail of theCD4 and causes CD4 degradation (Bour et al., Virology 69 (3):1510-20[1995]).

Therefore, in a preferred embodiment, the exogenous sequence expresses aVpu with reduced activity, and the Vpu has a reduced ability to triggerCD4 degradation.

The relevant biological activity of the well-characterized Tat proteinis the transactivation of transcription via interaction with thetransactivation response element (TAR). It was demonstrated that Tat isable to transactivate heterologous promoters lacking HIV sequences otherthan TAR (Han P. et al., Nucleic Acid Res 19 (25):7225-9 [1991]).

Thus, in a preferred embodiment, the exogenous sequence expresses a tatwith reduced activity, and the tat shows reduced transactivation ofpromoters via the TAR element.

In another preferred embodiment, the exogenous sequence expresses atransdominant Tat, and the transdominant Tat can be obtained by makingthe following substitutions: 22 (Cys>Gly) and 37 (Cys>Ser).

Nef protein is essential for viral replication responsible for diseaseprogression by inducing the cell surface downregulation of CD4 (Lou T etal., J Biomed Sci 4(4):132 [1997]). This downregulation is initiated bydirect interaction between CD4 and Nef (Preusser A. et al., BiochemBiophys Res Commun 292 (3):734-40 [2002]). Thus, Nef protein withreduced function shows reduced interaction with CD4.

Thus, in preferred embodiments, the exogenous sequence expresses a Nefwith reduced activity, and the Nef is truncated at the amino terminussuch as, for example, a Nef in which the 19 N-terminal amino acids aredeleted.

The relevant function of Rev is the posttranscriptional transactivationinitiated by interaction with the Rev-response element (RRE) of viralRNA (Iwai et al., Nucleic Acids Res 20 (24):6465-72 [1992]).

Thus, in a preferred embodiment, the exogenous sequence expresses a Revwith reduced activity, and the Rev shows a reduced interaction with theRRE.

In certain embodiments, the accessory/regulatory proteins are expressedindividually.

In other embodiments, multiple polypeptides are expressed and some orall of the polypeptides are expressed as fusion proteins. In thiscontext reference is made to WO 03/097675, the content of which isherewith incorporated by reference.

In a preferred embodiment, the recombinant MVA virus according to theinvention, such as MVA-BN and its derivatives, expresses a fusionprotein comprising at least four HIV polypeptides selected from thegroup consisting of the full-length proteins Vif, Vpr, Vpu, Vpx, Rev,Tat and Nef; and derivatives or parts thereof.

In another embodiment, the recombinant MVA virus according to theinvention, such as MVA-BN and its derivatives, expresses at least eightHIV polypeptides selected from the group consisting of the full-lengthVif, Vpr, Vpu, Rev, Tat, Nef, Gag and Pol; and derivatives or partsthereof.

In another embodiment, a recombinant MVA virus according to theinvention, such as MVA-BN and its derivatives, expresses one or morepolypeptides selected from the group consisting of:

-   -   (i) a fusion protein comprising Vif-Vpu-Vpr-Rev, ligated in this        order, or in a different order, wherein Vif, Vpu, Vpr and Rev        stand for full-length proteins, or parts or derivatives of the        full-length proteins;    -   (ii) a Nef, or a part or derivative thereof, in particular a Nef        protein in which N-terminal amino acids are deleted, such as the        first 19 amino acids;    -   (iii) a Tat, or a part or derivative thereof, in particular a        transdominant Tat; and    -   (iv) a Gag-Pol fusion protein, wherein Gag and Pol stand for        full-length proteins, or parts or derivatives of the full-length        proteins.

The number of expression cassettes from which the HIV polypeptides areexpressed is not critical. In one embodiment, the HIV polypeptides areexpressed from two to five expression cassettes comprising a pluralityof the following:

-   -   (i) an expression cassette expressing a Vif-Vpu-Vpr-Rev fusion        protein, wherein Vif, Vpu, Vpr and Rev stand for either        full-length proteins, or parts or derivatives of the full-length        proteins; arranged in the exemplified order, or in a different        order;    -   (ii) an expression cassette expressing Nef or a part or        derivative thereof, in particular a Nef protein in which        N-terminal amino acids are deleted, such as a Nef lacking the        first 19 amino acids;    -   (iii) an expression cassette expressing Tat or a part or        derivative thereof, in particular a transdominant Tat; and    -   (iv) an expression cassette expressing a Gag-Pol fusion protein,        wherein Gag and Pol stand for either full-length proteins or        parts or derivatives of the full-length proteins; arranged in        the exemplified order, or in the reverse order (i.e., Pol-Gag).

In a preferred embodiment, the expression of heterologous nucleic acidsequence is preferably, but not exclusively, under the transcriptionalcontrol of a poxvirus promoter. An example of a suitable poxviruspromoter is the cowpox ATI promoter (see WO 03/097844, incorporatedherein by reference). In certain embodiments, the expression of eachexpression cassette is controlled by a different promoter. In otherembodiments, all expression cassettes are controlled by a copy of thesame promoter.

In one embodiment, the invention relates to a recombinant virus in whichall HIV expression cassettes, such as the four expression cassettesexemplified above, are controlled by a cowpox ATI promoter or aderivative thereof, as defined in WO 03/097844.

In one embodiment, the expression cassettes can be inserted into 1 to 10insertion sites in the genome of a recombinant MVA according to theinvention, such as MVA-BN and its derivatives.

It was found that recombinant MVA viruses, in particular MVA-BN and itsderivatives, used for the expression of at least six HIV proteins, orsix parts or derivatives thereof, can be easily obtained if not allexpression cassettes are inserted into the same insertion side.

Thus, in certain embodiment, the different expression cassettes areinserted into 2 to 8, or 3 to 5, or into 3 insertion sites in the MVAgenome.

The insertion of heterologous nucleic acid sequence can be done into anon-essential region of the virus genome. According to one embodiment,the heterologous nucleic acid sequence is inserted at a naturallyoccurring deletion site of the MVA genome (disclosed in PCT/EP96/02926,incorporated herein by reference).

According to a further embodiment, one or more heterologous sequencescan be inserted into one or more intergenic regions of the MVA genome asdescribed herein.

Methods on how to insert heterologous sequences into the poxviral genomeare known to a person skilled in the art.

In a preferred embodiment, the expression cassettes comprising one ormore HIV proteins, parts or derivatives thereof, can be inserted intoone or more intergenic regions selected from the group consisting of IGR07-08, IGR I4L-I5L, and IGR 136-137 of the MVA genome, in particular thegenome of MVA-BN and its derivatives.

In another embodiment, the recombinant poxvirus is MVA-BN, or aderivative thereof, comprising all the following expression cassettesinserted into the specified insertion sites:

-   -   (i) an expression cassette expressing Vif-Vpu-Vpr-Rev as fusion        protein in this or a different order, wherein Vif, Vpu, Vpr and        Rev stand for either full-length proteins, or parts or        derivatives of the full-length proteins; inserted into the        intergenic region IGR 07-08;    -   (ii) a second expression cassette expressing Nef, or a part or        derivative thereof, in particular a Nef protein in which        N-terminal amino acids are deleted, such as Nef lacking the        first 19 amino acids; inserted into IGR I4L-I5L;    -   (iii) a third expression cassette that expresses Tat, or a part        or derivative thereof, in particular a transdominant Tat,        inserted into IGR 136-137; and    -   (iv) a fourth expression cassette that express a Gag-Pol fusion        protein, wherein Gag and Pot stand for either full-length        proteins or parts or derivatives of the full-length proteins;        inserted into IGR 136-137.

Thus, in the latter embodiment, the third and the fourth expressioncassettes are inserted into the same integration site. It is to be takeninto account that IGR I4L-I5L on the one side, and IGR 136-137 and IGR07-08 on the other side, belong to two different numbering systems;these numbering or nomenclature systems are explained above.

In a preferred embodiment, the recombinant virus according to theinvention can induce a protective immune response. The term “protectiveimmune response” as used herein is intended to mean that the vaccinatedsubject is able to control in some way an infection with the pathogenicagent against which the vaccination was done. Usually, the animal orsubject having developed a “protective immune response” develops milderclinical symptoms than an unvaccinated subject, and/or the progressionof the disease is slowed down.

The invention further relates to medicaments and vaccines comprising therecombinant MVA virus of the invention.

In other embodiments, the invention further relates to pharmaceuticalcompositions and vaccines comprising a recombinant MVA virus as definedabove.

In further embodiments, the invention further relates to the use of arecombinant MVA virus as defined above for the preparation of amedicament and/or vaccine for the treatment and/or prevention of AIDS.

In another embodiment, the invention further relates to a method ofprevention AIDS comprising the step of administration of a MVA virus asdefined above.

It is pointed out that the term “prevention of AIDS” as used in thecontext of the invention does not mean that the recombinant MVA virusprevents AIDS in all subjects under all conditions. To the contrary,this term as used herein refers to any statistically significantprotective effect, even if this effect is considered low.

Possible concentrations and modes of administration are indicated above.

Numerous ways to prepare recombinant MVA formulations are known to theskilled artisan, as are modes of storage. In this context, reference ismade to WO 03053463, incorporated herein by reference.

In a further embodiment, the invention relates to a host cell infectedwith a recombinant MVA virus as defined above. The host cell can be acell that is not part of an entire living organism.

In another embodiment, the invention further relates to the genome of arecombinant MVA virus according to the invention.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the appended claims.

EXAMPLES

The following examples will further illustrate the invention. It will beunderstood by any person skilled in the art that the provided examplesin no way are to be interpreted in a way that limits the invention tothese examples. The scope of the invention is only to be limited by thefull scope of the appended claims.

Example 1 Insertion Vectors pBNX39, pBNX70 and pBN84

For the insertion of exogenous sequences into the intergenic regionadjacent to the 065L ORF (insertion site is at genome position 56760) ofMVA, a vector was constructed which comprises about 1200 bp of theflanking sequences adjacent to the insertion site. These flankingsequence are separated into two flanks comprising, on one flank about610 bp of the 065L ORF (alternative nomenclature: I4L ORF), and on theother flank about 580 bp of the intergenic region beHind the 065L ORF,as well as parts of the proximate ORF. In between these flankingsequences, there is an Ecogpt gene (gpt stands forphosphoribosyltransferase gene isolated from E. coli) and a BFP (bluefluorescence protein), respectively, under the transcriptional controlof a poxviral promoter. Additionally, there is at least one cloning sitefor the insertion of additional genes or sequences to be inserted intothe intergenic region beHind the I4L ORF. Exemplary vector constructsaccording to the invention are disclosed in FIGS. 1A and 1B (pBNX39,pBNX70). In vector pBN84 (FIG. 1C) the coding region for Dengue virusNS1 is inserted in the cloning site of pBNX70 (FIG. 1B).

Generation of the Recombinant MVA via Homologous Recombination

Foreign genes can be inserted into the MVA genome by homologousrecombination. For that purpose the foreign gene of interest is clonedinto a plasmid vector, as described above. This vector is transfected inMVA infected cells. The recombination takes place in the cytoplasm ofthe infected and transfected cells. With help of the selection and/orreporter cassette, which is also contained in the insertion vector,cells comprising recombinant viruses are identified and isolated.

For homologous recombination BHK (Baby hamster kidney) cells or CEF(primary chicken embryo fibroblasts) are seeded in 6 well plates usingDMEM (Dulbecco's Modified Eagles Medium, Gibco BRL) containing 10% fetalcalf serum (FCS), or VP-SFM (Gibco BRL) containing 4 mmol/l L-Glutaminefor a serum free production process.

Cells need to be still in the growing phase and therefore should reach60-80% confluence on the day of transfection. Cells are counted beforeseeding, as the number of cells has to be known for determination of themultiplicity of infection (moi) for infection.

For the infection, the MVA stock is diluted in DMEM/FCS orVP-SFM/L-Glutamine so that 500 μl dilution contain an appropriate amountof virus that will give a moi of about 0.1-1.0. Cells are assumed tohave divided once after seeding. The medium is removed from cells andcells are infected with 500 μl of diluted virus for 1 hour rocking atroom temperature. The inoculum is removed and cells are washed withDMEM/VP-SFM. Infected cells are left in 1.6 ml DMEM/FCS orVP-SFM/L-Glutamine, respectively, while setting up the transfectionreaction (Qiagen Effectene Kit).

For the transfection, the “Effectene” transfection kit (Qiagen) is used.A transfection mix is prepared of 1-2 μg of linearized insertion vector(total amount for multiple transfection) with buffer EC to give a finalvolume of 100 μl. Then, 3.2 μl of Enhancer are added, the mixturevortexed and incubated at room temperature for 5 min. Then, 10 μl ofEffectene are added after vortexing the stock tube, the solution ismixed thoroughly by vortexing, and incubated at room temperature for 10min. Next, 600 μl of DMEM/FCS or VP-SFM/L-Glutamine, respectively, areadded, mixed and subsequently, the whole transfection mix is added tothe cells, which are already covered with medium. The dish is rockedgently to mix the transfection reaction. The incubation takes place at37° C. with 5% CO₂ overnight. The next day, the medium is removed andreplaced with fresh DMEM/FCS or VP-SFM/L-Glutamine. Incubation iscontinued until day 3.

For harvesting, the cells are scraped into medium, and then the cellsuspension is transferred to an adequate tube and frozen at −20° C. forshort-term storage, or at −80° C. for long-term storage.

Insertion of Ecogpt in the I4L Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBNX39 (FIG. 1A) containing the Ecogpt gene (Ecogpt, orshortened to gpt, stands for phosphoribosyltransferase gene) as areporter gene. Resulting recombinant viruses were purified by 3 roundsof plaque purification under phosphribosyl-transferase metabolismselection by addition of mycophenolic acid, xanthin, and hypoxanthin.Mycophenolic acid (MPA) inhibits inosine monophosphate dehydrogenase,and results in blockage of purine synthesis and inhibition of viralreplication in most cell lines. This blockage can be overcome byexpressing Ecogpt from a constitutive promoter and providing thesubstrates xanthine and hypoxanthine.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the I4L insertion side, the primer pair comprising BN499 (CAACTC TCT TCT TGA TTA CC, SEQ ID NO.:1) and BN500 (CGA TCA AAG TCA ATC TATG, SEQ ID NO.:2) was used. When the DNA of the empty vector virus MVA isamplified, the expected PCR fragment is 328 nucleotides (nt) long.However, when a recombinant MVA is amplified, which has incorporatedexogenous DNA at the I4L insertion site, the fragment is correspondinglyenlarged.

Insertion of NS1 in the IGR064L-065L (I4L-I5L) Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol, and were additionally transfected withinsertion vector pBN84 (FIG. 1C) containing the Ecogpt gene forselection and BFP (Blue fluorescence protein) as a reporter gene.Resulting recombinant viruses were purified by 7 rounds of plaquepurification under phosphribosyl-transferase metabolism selection byaddition of mycophenolc acid, xanthin and hypoxanthin.

Resulting recombinant viruses were identified by standard PCR assays,using a primer pair selectively amplifying the expected insertion site.To amplify the I4L-15L insertion site, the primer pair comprising BN499(CAA CTC TCT TCT TGA TTA CC, SEQ ID NO.:1) and BN500 (CGA TCA AAG TCAATC TAT G, SEQ ID NO.:2) were used. When the DNA of the empty vectorvirus MVA is amplified, the expected PCR fragment is 328 nucleotides(nt) long. When a recombinant MVA for NS1 is amplified, which hasincorporated Dengue virus NS1 coding region at the I4L insertion site,the PCR fragment is expected to be 1683 bp. The PCR results in FIG. 7show clearly the stable insertion of NS1 in the I4L insertion site after17 rounds of virus amplification.

Testing of recMVA Including NS1 (MVA-BN22) In Vitro

T25 flasks with about 80% confluent monolayers of BHK cells wereinoculated with 100 μl of the virus stock diluted to 1×10⁷ in MEMacontaining 1% FCS, and rocked at room temperature for 30 minutes. 5 mlof MEMa containing 3% FCS were added to each flask and incubated at 30°C. in a CO₂ incubator. The flasks were harvested after 48 hours. Thesupernatant was removed from each flask, and spun at 260×g for 10minutes at 4° C. The resulting supernatants were stored in aliquots at−80° C. The pellets were each washed with 5 ml of 1×PBS twice and thenresuspended in 1 ml of hypotonic douncing buffer containing 1% TritonX100. The cell lysates were harvested and spun for 5 minutes at16,000×g, and the resulting supernatants were stored in microcentrifugetubes at −80° C.

Flasks inoculated with MVA including GFP, MVA including the NS1 geneinserted in a deletion site (MVA-BN07), and mock infected flasks werealso treated the same way as described above.

The cell/viral lysate and the supernatant were treated innon-reducing/reducing sample buffer under non-heated/heated conditions,respectively. The proteins were separated by 10% SDS PAGE andtransferred to nitrocellulose membranes. The blots were probed overnightwith pooled convalescent patients' sera (PPCS) at 1:500 dilution. Afterwashing 3 times with 1×PBS, the blots were incubated with anti-humanIgG-HRP (DAKO) for 2 hours at room temperature. After the blots werewashed as described before, the color was developed using 4chloro-1-naphtol.

The western blot results showed that NS1 in MVA-BN22 is expressed inlarge quantities. NS1 was expressed in the right conformation, i.e., asa dimer under non-heated/non-reducing conditions, and as a monomer underheated/reducing conditions.

The NS1 expression was compared in both MVA-BN22 and MVA-BN07. The BHKcells were inoculated with the same pfu and harvested after 48 hours.The results showed that the expression of NS1 was much higher in BN22than in BN07. The western blots results also showed that there is moreNS1 secreted in the supernatant with the BN22 construct compared toBN07.

The results also showed that NS1 expressed in cells infected with BN22is antigenic and is recognized by the pooled convalescent patients'sera.

In conclusion, NS1 is expressed in large quantities and in the rightconfirmation in the BHK cells infected with BN22. Both the dimer andmonomer are antigenic, and are recognized by the pooled convalescentpatients' sera.

Example 2 Insertion Vectors pBNX67 and pBN27

The MVA sequences adjacent to the new insertion site (at genome position129940) between the ORF 136L and 137L were isolated by standard PCRamplification of the sequence of interest using the following primers:

oBN543 (TCCCCGCGGAGAGGCGTAAAAGTTAAATTAGAT; SEQ ID NO.: 3) and oBN544(TGATCTAGAATCGCTCGTAAAAACTGCGGAGGT; SEQ ID NO.: 4) for isolating Flank1; oBN578 (CCGCTCGAGTTCACGTTCAGCCTTCATGC; SEQ ID NO.: 5) and oBN579(CGGGGGCCCTATTTTGTATAATATCTGGTAAG; SEQ ID NO.: 6) for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and XbaI, and ligated to a SacII/XbaI-digested anddephosphorylated basic vector, pBluescript (Stratagene).

The resulting plasmid was XhoI/ApaI-digested, dephosphorylated andligated to the XhoI/ApaI-digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers oBN545 (CGGCTGCAGGGTACCTTCACGTTCAGCCTTCATGC; SEQID NO.:7) and oBN546 (CGGAAGCTTTATATGGTTTAGGATATTCTGTTTT; SEQ ID NO.:8),and which became HindIII/PstI-digested, was inserted into theHindIII/PstI site of the resulting vector. FIG. 2A shows the resultingvector (pBNX51).

A reporter cassette comprising a synthetic promoter, NPT II gene(neomycin resistance), poly-A region, IRES, and EGFP gene(Ps-NPTII-polyA-IRES-EGFP) was EcI136II/XhoI-digested and inserted intothe HindIII/XhoI site of the insertion vector, wherein the HindIII sitewas blunt ended with T4 DNA Polymerase (Roche). A restriction map of anexemplary vector construct according to this example is disclosed inFIG. 2B (pBNX67).

For construction of pBN27 (FIG. 2C) the Dengue virus PrM of serotype 4was inserted in the single PacI site of pBNX67.

Generation of the Recombinant MVA via Homologous Recombination

The vector pBNX67 (FIG. 2B) can be used to generate a recombinant MVAusing the above mentioned protocol. For example, using pBN27 (FIG. 2C)for homologous recombination results in a recombinant MVA carryingDengue virus PrM4 in the intergenic region between two adjacent ORFs.

Insertion of PrM4 in the IGR136-137 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol, and were additionally transfected withinsertion vector pBN27 (FIG. 2C) containing the NPT gene for selection,and EGFP (enhanced green fluorescence protein) as a reporter gene.Resulting recombinant viruses were purified by 4 rounds of plaquepurification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR 136-137 insertion site, the primer pair comprisingBN900 (cgttcgcatgggttacctcc, SEQ ID NO.:9) and BN901(gacgcatgaaggctgaac, SEQ ID NO.:10) was used. When the DNA of the emptyvector virus MVA is amplified the expected PCR fragment is 88nucleotides (nt) long. When a recombinant MVA for PrM4 is amplified,which has incorporated Dengue virus PrM4 coding region at the IGR136-137insertion site, the fragment is expected to be 880 bp. The PCR resultsin FIG. 8A show clearly the stable insertion of PrM4 in the IGR136-137insertion site after 22 rounds of virus amplification. The recombinantMVA still shows the same growth characteristics as MVA-BN. It replicatesin chicken embryo fibroblasts (CEF cells) and grows attenuated inmammalian cells (FIG. 8B).

Example 3 Insertion vectors pBNX79, pBNX86, pBNX88, pBN34 and pBN56

The MVA sequences adjacent to the new insertion site (at genome position12800) between the ORF 007R and 008L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR 07/08 F1up (CGCGAGCTCAATAAAAAAAAGTTTTAC; SEQ ID NO.: 11) and IGR07/08 F1end (AGGCCGCGGATGCATGTTATGCAAAATAT; SEQ ID NO.: 12) forisolating Flank 1; IGR 07/08 F2up (CCGCTCGAGCGCGGATCCCAATATATGGCATAGAAC;SEQ ID NO.: 13) and IGR 07/08 F2end (CAGGGCCCTCTCATCGCTTTCATG; SEQ IDNO.: 14) for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and SacI, and ligated to a SacII/SacI-digested anddephosphorylated pBluescript plasmid (Stratagene).

The resulting plasmid was XhoI/ApaI-digested, dephosphorylated andligated to the XhoI/ApaI-digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers IGR 07/08 F2up(CCGCTCGAGCGCGGATCCCAATATATGGCATAGAAC; SEQ ID NO.:13) and IGR 07/08F2mid (TTTCTGCAGTGATATTTATCCAATACTA; SEQ ID NO.:15), and which isBamHI/PstI digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutic gene comprising cassette, having for examplea poxviral promoter, a marker gene, a poly-A region and optionally anIRES element, a further gene, for example, one expressing atherapeutically active substance or gene product, can be blunt-endedwith T4 DNA Polymerase (Roche) after a restriction digest, and insertedinto a suitable cloning site of the plasmid vector. A restriction map ofan exemplary vector construct according to this example is disclosed inFIG. 3A (pBNX79). Insertion of the NPT/EGFP selection cassette resultedin vector pBNX86 (FIG. 3B) and insertion of the gpt/BFP selectioncassette resulted in vector pBNX88 (FIG. 3C). Considering an expressionunit for a therapeutic gene, comprising a therapeutic gene and anoperably linked promoter, this expression unit is inserted into the PacIsite. For construction of pBN34 (FIG. 3D), the Dengue virus PrM2 wascloned in pBNX88 (FIG. 3C); and for synthesis of pBN56 (FIG. 3E) the HIVenv coding region was cloned into the PacI site of pBNX86 (FIG. 3B).

Generation of the Recombinant MVA via Homologous Recombination

The vectors pBNX86 (FIG. 3B) and pBNX88 (FIG. 3C), respectively, can beused to generate a recombinant MVA using the above mentioned protocol.Using pBN34 (FIG. 3D) for homologous recombination results in arecombinant MVA carrying Dengue virus PrM2 in the intergenic regionbetween two adjacent ORFs. Recombination of pBN56 (FIG. 3E) with theMVA-BN genome results in a recombinant MVA, which contains the HIV envgene in the corresponding IGR.

Insertion of PrM2 in the IGR07-08 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol, and were additionally transfected withinsertion vector pBN34 (FIG. 3D) containing the gpt gene for selectionand BFP as reporter gene. Resulting recombinant viruses were purified by3 rounds of plaque purification under selection by mycophenolic acid, asdescribed in Example 1.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR 07-08 insertion site, the primer pair comprisingBN902 (ctggataaatacgaggacgtg, SEQ ID NO.:16) and BN903(gacaattatccgacgcaccg, SEQ ID NO.:17) was used. When the DNA of theempty vector virus MVA is amplified, the expected PCR fragment is 190nucleotides (nt) long. When a recombinant MVA for PrM2 is amplified,which has incorporated Dengue virus PrM2 coding region at the IGR 07-08insertion site, the fragment is expected to be 950 bp. The PCR resultsin FIG. 9A show clearly the stable insertion of PrM2 in the IGR 07-08insertion site after 20 rounds of virus amplification. The recombinantMVA still shows the same growth characteristics as MVA-BN. It replicatesin chicken embryo fibroblasts (CEF cells), and grows attenuated inmammalian cells (FIG. 9B).

Insertion of HIV env in the IGR07-08 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN56 (FIG. 3E) containing the NPT gene for selectionand EGFP as reporter gene. Resulting recombinant viruses were purifiedby 6 rounds of plaque purification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IR 07-08 insertion site, the primer pair comprising BN902(ctggataaatacgaggacgtg, SEQ ID NO.:16) and BN903 (gacaattatccgacgcaccg,SEQ ID NO.:17) was used. When the DNA of the empty vector virus MVA isamplified the expected PCR fragment is 190 nucleotides (nt) long. When arecombinant MVA for env is amplified, which has incorporated HIV envcoding region at the IGR 07-08 insertion site, the fragment is expectedto be 2.6 kb. The PCR results in FIG. 10 show clearly the stableinsertion of env in the IGR 07-08 insertion site after 20 rounds ofvirus amplification.

Example 4 Insertion vector pBNX80, pBNX87 and pBN47

The MVA sequences adjacent to the new insertion site (at genome position37330) between the ORF 044L and 045L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR44/45F1up (CGCGAGCTCATTTCTTAGCTAGAGTGATA; SEQ ID NO.: 18) andIGR44/45F1end (AGGCCGCGGAGTGAAAGCTAGAGAGGG; SEQ ID NO.: 19) forisolating Flank 1; IGR44/45F2up (CCGCTCGAGCGCGGATCCTAAACTGTATCGATTATT;SEQ ID NO.: 20) and IGR44/45F2end (CAGGGCCCCTAAATGCGCTTCTCAAT; SEQ IDNO.: 21) for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and SacI, and ligated to a SacII/SacI-digested anddephosphorylated basic vector, pBluescript (Stratagene).

The resulting plasmid was XhoI/ApaI digested, dephosphorylated andligated to the XhoI/ApaI-digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers IGR44/45F2up(CCGCTCGAGCGCGGATCCTAAACTGTATCGATTATT; SEQ ID NO.:20) and IGR44/45F2mid(TTTCTGCAGCCTTCCTGGGTTTGTATTAACG; SEQ ID NO.:22), and which becameBamHI/PstI-digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutical gene comprising cassette, having forexample a poxviral promoter, a marker gene, a poly-A region andoptionally an IRES element, a further gene, for example expressing atherapeutically active substance or gene product, can be blunt endedwith T4 DNA Polymerase (Roche) after a restriction digest, and insertedinto a suitable cloning site of the plasmid vector. Considering areporter gene cassette, the PstI, EcoRI, EcoRV, HindIII, AvaI, or XhoIrestriction enzyme site between Flank 2 and the Flank-2-repetition ispreferred as a cloning site. For the construction of pBNX87 (FIG. 4B),the NPT/EGFP selection cassette was inserted in pBNX80 (FIG. 4A).Considering an expression unit for a therapeutic gene, comprising atherapeutic gene and an operably linked promoter, this expression unitis inserted into the PacI site.

Restriction maps of exemplary vector constructs according to thisexample are disclosed in FIGS. 4A and 4B (pBNX80, pBNX87).

The vector can be used to generate a recombinant MVA, following theabove-mentioned protocol, carrying an exogenous sequence in theintergenic region between two adjacent ORFs.

For the construction of pBN47 (FIG. 4C), the PrM of Dengue virusserotype 3 was cloned into pBNX87 (FIG. 4B).

Insertion of PrM3 in the IGR44-45 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol, and were additionally transfected withinsertion vector pBN47 (FIG. 4C), containing the NPT gene for selectionand EGFP as reporter gene. Resulting recombinant viruses were purifiedby 3 rounds of plaque purification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR 44-45 insertion site, the primer pair comprisingBN904 (cgttagacaacacaccgacgatgg, SEQ ID NO.:23) and BN905(cggatgaaaaatttttggaag, SEQ ID NO.:24) was used. When the DNA of theempty vector virus MVA is amplified, the expected PCR fragment is 185nucleotides (nt) long. When a recombinant MVA for PrM3 is amplified,which has incorporated Dengue virus PrM3 coding region at the IGR 44-45insertion site, the fragment is expected to be 850 bp. The PCR resultsin FIG. 11A show clearly the stable insertion of PrM3 in the IGR44-45insertion site after 19 rounds of virus amplification. The recombinantMVA (MVA-mBN28) still shows the same growth characteristics as MVA-BN.It replicates in chicken embryo fibroblasts (CEF cells) and growsattenuated in mammalian cells (FIG. 11B).

Example 5 Insertion Vectors pBNX90, pBNX92 and pBN54

The MVA sequences adjacent to the new insertion site (at genome position137496) between the ORFs 148R and 149L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR148/149F1up (TCCCCGCGGGGACTCATAGATTATCGACG; SEQ ID NO.: 25) andIGR148/149F1end (CTAGTCTAGACTAGTCTATTAATCCACAGAAATAC; SEQ ID NO.: 26)for isolating Flank 1; IGR148/149F2up(CCCAAGCTTGGGCGGGATCCCGTTTCTAGTATGGGGATC; SEQ ID NO.: 27) andIGR148/149F2end (TAGGGCCCGTTATTGCCATGATAGAG; SEQ ID NO.: 28) forisolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and XbaI, and ligated to a SacII/XbaI-digested anddephosphorylated basic vector, pBluescript (Stratagene).

The resulting plasmid was HindIII/ApaI digested, dephosphorylated andligated to the HindIII/ApaI-digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers IGR148/149F2up(CCCAAGCTTGGGCGGGATCCCGTTTCTAGTATGGGGATC; SEQ ID NO.:27) andIGR148/149F2mid (TTTCTGCAGTGTATAATACCACGAGC; SEQ ID NO.:29) and whichbecame BamHI/PstI-digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutic gene comprising cassette, having for examplea poxviral promoter, a marker gene, a poly-A region, and optionally anIRES element, a further gene, for example, a gene expressing atherapeutically active substance or gene product, can be blunt endedwith T4 DNA Polymerase (Roche) after a restriction digest and insertedinto a suitable cloning site of the plasmid vector. For construction ofpBNX92 (FIG. 5B), the gpt/BFP expression cassette was inserted in thiscloning site. Considering a reporter gene cassette, the PstI, EcoRI,EcoRV, and HindIII restriction enzyme site between Flank 2 and theFlank-2-repetition is preferred as a cloning site. Considering anexpression unit for a therapeutic gene, comprising a therapeutic geneand an operably linked promoter, this expression unit is inserted intothe PacI site. For construction of pBN54 (FIG. 5C) the Dengue virus PrM1was inserted in this PacI site.

Restriction maps of exemplary vector constructs according to thisExample are disclosed in FIGS. 5A and 5B (pBNX90, pBNX92).

The vector can be used to generate a recombinant MVA, following theabove-mentioned protocol, carrying an exogenous sequence in theintergenic region between two adjacent ORFs. For the generation of arecombinant MVA expressing the Dengue virus PrM1, pBN54 (FIG. 5C) wasused for a homologous recombination.

Insertion of PrM1 in the IGR148-149 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN54 (FIG. 5C) containing the gpt gene for selectionand BFP as reporter gene. Resulting recombinant viruses were purified by3 rounds of plaque purification under selection with mycophenolic acid.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR148-149 insertion site, the primer pair comprisingBN960 (ctgtataggtatgtcctctgcc, SEQ ID NO.:30) and BN961(gctagtagacgtggaaga, SEQ ID NO.:31) was used. When the DNA of the emptyvector virus MVA is amplified the expected PCR fragment is 450nucleotides (nt) long. When a recombinant MVA for PrM1 is amplified,which has incorporated Dengue virus PrM1 coding region at the IGR148-149insertion site, the fragment is expected to be 1200 bp. The PCR resultsin FIG. 12 a show clearly the stable insertion of PrM1 in the IGR148-149insertion site after 23 rounds of virus amplification. The recombinantMVA still shows the same growth characteristics as MVA-BN, namely, itreplicates in chicken embryo fibroblasts (CEF cells) and growsattenuated in mammalian cells (FIG. 12 b).

Example 6 Generation of a Recombinant MVA-BN Comprising in the ViralGenome a Truncated nef Gene, a gag-pol Fusion Gene, a Transdominant TatGene and a Vif-Vpr-Vpu-Rev Fusion Gene, each Under the Control of theATI Promoter

Origin of the HIV Genes Delta nef, gag-pol, vif-vpr-vpu-rev and tat

The Gag-pol fused gene was obtained by PCR from DNA from HXB2 infectedcells. The Nef gene was amplified by PCR from DNA of MVA-nef (LAI) toobtain a truncated version. The first 19 aa were deleted resulting inNef-truncated. The Vif and Vpu genes were generated by RT-PCR from HIVRNA from a primary isolate MvP-899, while the Vpr, Rev and Tat geneswere synthesized by oligo annealing based on the sequence of HXB2. Theprotein tat-mutated was created by introducing two mutations in tat,which are not localized in important epitopes but lead to the loss oftransactivating activity. The mutations are the following substitutions:22 (Cys>Gly) and 37 (Cys>Ser).

The DNA constructs were cloned into recombinant vectors.

Transfer Vectors pBNX59, pBNX67 and pBNX86

pBNX59, pBN67 and pBNX86 are plasmid vectors containing MVA DNAsequences that are homologous to intergenic regions. When an expressioncassette is inserted into such an MVA sequence in a plasmid, it ispossible to use the resulting plasmid for homologous recombination ofthe expression cassettes into the homologous intergenic non-codingregion of the MVA genome. pBNX59 directs homologous recombination intoIGR I4L-I5L; pBNX67 (see example 2) directs homologous recombinationinto IGR 136-137, and pBNX86 (see example 3) directs homologousrecombination into IGR 07-08 of the MVA genome.

MVA DNA sequences spanning the intergenic regions between coding regionsI4L and I5L (i.e. IGR I4L-I5L), 136 and 137 (i.e. IGR 136-137), and 07and 08 (i.e. IGR 07-08) in the HindIII I fragment of the MVA genome,were amplified and cloned into pBluescript K S+. The coding sequence forthe E. coli gpt gene under the control of a strong synthetic Vacciniavirus promoter was inserted between I4I and I5L flanks to generate theplasmid pBNX59.

The coding sequences for NPTII and a further marker gene (EGFP) underthe control of a vaccinia virus promoter (Ps) were inserted between 136and 137 flanks of MVA DNA to generate the recombination vector pBNX67(for details, see FIG. 2B and Example 2) that allows the selection ofrecombinant viruses.

The coding sequences for NPTII and a further marker gene (EGFP) underthe control of a strong Vaccinia virus promoter (P) were insertedbetween 07 and 08 flanks of MVA DNA to generate the plasmid pBNX86 (fordetails, see FIG. 3B and Example 3).

Sequence repeats of flank 2 were inserted in order to allow the deletionof the selection cassettes after isolation of recombinant viruses.

Cloning and Generation of MVA-mBN87B Comprising in the Viral Genome aTruncated nef Gene, a gag-pol Fusion Gene, a Transdominant Tat Gene anda Vif-Vpr-Vpu-Rev Fusion Gene, each Under the Control of the ATIPromoter

To create a recombinant MVA-BN strain which expresses the severalmultiantigen constructs, the recombination plasmids pBN108, pBN131 andpBN175 were created (see below).

Plasmid pBN108 (FIG. 13A) was obtained as described below. It comprisesa gag-pol fusion gene and a transdominant Tat, each under the control ofthe ATI promoter. pBN108 was then transfected into primary CEF cellsinfected with mBN67B. The double recombinant MVA virus mBN78A, whichcarried gpt, a further marker gene, truncated nef, and the transdominanttat and gag-pol fusion coding region, was obtained after multiple plaquepurifications under selective conditions of recombinant MVA fromfluorescing plaques. Then, the selection cassette was deleted, byleaving out selection conditions, and mBN78B was obtained.

pBN131 (FIG. 13B), obtained as described below, was transfected intoprimary CEF cells infected with MVA-BN. The resulting recombinant MVAvirus mBN67A, which carried the NPTII, a further marker gene (EGFP), andthe truncated nef coding region, was obtained after multiple plaquepurifications, under selective conditions of recombinant MVA, fromfluorescing plaques (the fluorescence being conferred by the presence ofthe EGFP marker). Then, the selection cassette was deleted, by leavingout selection conditions, and mBN67B was obtained.

In parallel, plasmid pBN175 (FIG. 13C) was generated as described below.It comprises the vif-vpr-vpu-rev coding region under the control of theATI promoter. pBN175 was then transfected into primary CEF cellsinfected with mBN78B. The triple recombinant MVA virus mBN87A, whichcarried a marker gene and gpt and the vif-vpr-vpu-rev coding region, wasobtained after multiple plaque purifications under selective conditionsof recombinant MVA from fluorescing plaques. After amplification andplaque purification under non-selective conditions the recombinant virusMVA-mBN87B, devoid of the selection cassettes, was isolated.

Cloning of the Recombination Plasmid pBN131 Comprising a Truncated NefGene Under the Control of the ATI Promoter

The nef gene was generated by PCR out of a recombinant MVA comprisingthe nef gene, and the purified PCR product was cloned into TOPO TAvector from Invitrogen. The first 19 amino acids were truncated by PCR,and the resulting truncated nef was restricted with BamHI and XhoI, andpurified by gel extraction. Vector pBNX65, which contains an ATIpromoter, was restricted with BamHI and XhoI, gel extracted anddephosphorylated, and ligated with the truncated nef. Positive cloneswere selected by HindIII digestion. The resulting plasmid pBN29, and therecombination vector pBNX59 (IGR I4L-I5L) were restricted with PacI; theinsert was gel purified, while the vector was dephosphorylated beforeligation. Positive clones of the resulting plasmid pBN31 were selectedby SalI and ScaI digestion. The ATI-truncated nef gene clone #11 wasused for homologous recombination.

Generation of a Recombinant MVA (mBN67A) Comprising a Truncated nef GeneUnder the Control of the ATI Promoter

Primary CEF cells were prepared in serum-free medium containing 4 mML-Glutamine and seeded in six-well plates. Cells were incubated for24-48 h until about 60 to 80% confluence. Cells were infected withMVA-BN at a MOI of 1.0. At about 60 min after infection, cells weretransfected with 0.5 μg of plasmid DNA (pBN131) per well using Effectene(Qiagen). The transfected cells were harvested after 2 days. The viruswas released by three cycles of freeze thawing. Tenfold dilutions of thevirus-containing supernatant were used to infect freshly prepared CEFcells at 90% confluence in VP-SFM serum-free medium containingL-Glutamine, and Xanthine, Hypoxanthine, and Mycophenolic acid (Sigma)were added. The cells were incubated for another 48 h.

To select recombinant single clones, cells were seeded in 96-well platesand tenfold dilutions of the virus-containing supernatant were used toinfect the cells. Plaque purification was performed after 48 h. Singlevirus-plaques detected in the highest dilution were selected, andharvested in VP-SFM. Virus-plaques were stored at −20° C., or directlyused for infection of new CEF cells.

After 4 rounds of plaque purification, the insertion of the foreign DNA(truncated nef) and absence of wild-type virus was confirmed by PCR. Theresulting recombinant virus clone was named mBN67A. After 2plaque-purifications under non selective conditions the recombinantvirus MVA-mBN67B, mostly devoid of the selection cassette, was isolated.

Cloning of the Recombination Plasmid pBN108 Comprising a gag-pol FusionGene and a Transdominant Tat, Each Under the Control of the ATI Promoter

The gag-pol fusion protein was generated by PCR including restrictionsites. The PCR product was restricted with BamHI and XhoI. This fragmentwas ligated to the BamHI and XhoI-restricted and dephosphorylated vectorpBNX65 (containing ATI promoter), resulting in pBN73. Positive pBN73clones were screened by XbaI restriction. To create a necessaryframeshift between gag and pol one nucleotide (T) was inserted,resulting in pBN97.

Positive pBN97 clones were screened by specific PCR for the fusion site.To insert transdominant tat into pBN97, tat was generated byoligoannealing and PCR including appropriate restriction sites, and wasinserted after restriction with Acc65I into pBNX65 (containing ATIpromoter), also restricted with Acc65I and dephosphorylated. To identifypositive clones of the resulting vector, preparations of DNA werescreened with HindIII. Further, to obtain transdominant tat, 2 aminoacid exchanges, at positions 22 (Cys>Gly) and 37 (Cys>Ser), were done,and the resulting vector pBN59 was obtained.

Next, transdominant tat was amplified out of pBN59 by PCR includingrestriction sites and ATI promoter. The PCR product was restricted withXbaI, and ligated to XbaI-restricted and dephosphorylated pBN97,resulting in pBN98. Positive right orientated clones were identified bydigest with Acc65I. ATI-td tat and ATI-gag-pol were restricted withPacI, gel extracted, and then ligated with PacI-linearized anddephosphorylated recombination vector pBNX67 (IGR 136-137), resulting inpBN108. Positive clones were selected by PacI restriction, and forproper orientation they were also checked by sequencing. The ATI-td tatand ATI-gag-pol positive clone #8 was used for homologous recombination.

Generation of a Recombinant MVA (mBN78A) Comprising a Truncated nefGene, a gag-pol Fusion Gene and a Transdominant Tat Gene, each Under theControl of the ATI Promoter

Primary CEF cells were prepared in serum-free medium containing 4 mML-Glutamine and seeded in six-well plates. Cells were incubated for24-48 h until about 60 to 80% confluence. Cells were infected withMVA-mBN67B at a MOI of about 1.0. About 60 min after infection, cellswere transfected with 0.5 μg plasmid DNA (pBN108) per well usingEffectene (Qiagen). The transfected cells were harvested after 2 days.Virus was released by three cycles of freeze thawing. Tenfold dilutionsof the virus-containing supernatant were used to infect freshly preparedCEF cells at 90% confluence, VP-SFM serum-free medium containingL-Glutamine and G418 (Gibco/Invitrogen) was added, and the cells wereincubated for another 48 h.

To select recombinant single clones, cells were seeded in 96-well platesand tenfold dilutions of the virus containing supernatant were used toinfect the cells. Plaque purification was performed after 48 h. Singlevirus-plaques detected in the highest dilution plates were selected, andharvested in VP-SFM. Virus-plaques were stored at −20° C., or directlyused for infection of new CEF cells.

After 5 rounds of plaque purification, the insertion of the foreign DNAand absence of wild-type virus was confirmed by PCR. The resultingrecombinant virus clone was named mBN78A. After 4 plaque-purificationsunder non selective conditions the recombinant virus MVA-mBN78 B, mostlydevoid of the selection cassette, was isolated.

Cloning of the Recombination Plasmid pBN175 Comprising a Vif-Vpr-Vpu-RevFusion Gene Under the Control of the ATI Promoter

The accessory genes vif, vpr, vpu and rev were generated as a fusionprotein by oligoannealing followed by PCR, and included restrictionsites within the primers. After restriction of the PCR products withBamHI and XhoI, the genes were inserted into the BamHI/XhoI-restricted,purified and dephosphorylated pBNX65 vector (containing ATI promoter),resulting in pBN32. Clones were analysed by BamHI restriction andsequencing. Five mutations noticed in the vif-vpr-vpu-rev sequence werecorrected by in vitro mutagenesis. The corrected pBN32 was thenrestricted with KpnI, blunted with T4-polymerase, and then cut withNotI. The recombination vector pBNX86 (IGR 07/08) was restricted withPacI, blunted and restricted with NotI, and then ligated withATI-vif-vpr-vpu-rev. Positive clones of the resulting plasmid pBN175were selected by restriction analysis with SphI.

Generation of a Recombinant MVA (mBN87B) Comprising a Truncated nefGene, a gag-pol Fusion Gene, a Transdominant Tat Gene, and aVif-Vpr-Vpu-Rev Fusion Gene, Each Under the Control of the ATI Promoter

Primary CEF cells were prepared in serum-free medium containing 4 mML-Glutamine and seeded in six-well plates. Cells were incubated for24-48 h until about 60 to 80% confluence. The cells were infected withMVA-mBN78B at a MOI of about 1.0. At 60 min after infection cells weretransfected with 0.5 μg of plasmid DNA (pBN175) per well using Effectene(Qiagen). The transfected cells were harvested after 2 days. Virus wasreleased by three cycles of freeze thawing. Tenfold dilutions of thevirus-containing supernatant were used to infect freshly prepared CEFcells at about 90% confluence. VP-SFM serum-free medium containingL-Glutamine and G418 (Gibco BRL) was added, and the cells were incubatedfor another 48 h.

To select recombinant single clones, cells were seeded in 96-well platesand tenfold dilutions of the virus-containing supernatant were used toinfect the cells. Plaque purification was performed after 48 h. Singlevirus-plaques detected in the highest dilution plates were selected, andharvested in VP-SFM. Virus-plaques were stored at −20° C., or directlyused for infection of new CEF cells.

After 5 rounds of plaque purification, the insertion of the foreign DNA(truncated nef gene, a gag-pol fusion gene, a transdominant Tat gene,and a Vif-Vpr-Vpu-Rev fusion gene) and absence of wild-type virus wasconfirmed by PCR. The resulting recombinant virus clone was namedmBN87A. After 5 plaque-purifications under non selective conditions therecombinant virus MVA-mBN87 B devoid of the selection cassette could beisolated. The identity of the recombinant vector was confirmed bystandard methods.

1. A recombinant modified vaccinia Ankara (MVA) virus comprising one ormore human immunodeficiency virus (HIV) DNA coding sequences insertedinto one or more intergenic regions (IGRs) of the viral genome, whereinthe IGR is selected from the group consisting of IGRs: 001L-002L,002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L, 017L-018L,018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L, 025L-026L,028R-029L, 030L-031L, 031L-032L, 032L-033L, 035L-036L, 036L-037L,037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R, 049L-050L,050L-051L, 051L-052R, 052R-053R, 053R-054R, 054R-055R, 055R-056L,061L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R, 078R-079R,080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R, 088R-089L,089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R, 101R-102R,103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L, 110L-111L,113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R, 122R-123L,123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R, 137L-138L,141L-142R, 143L-144R, 144R-145R, 145R-146R, 146R-147R, 147R-148R,148R-149L, 152R-153L, 153L-154R, 154R-155R, 156R-157L, 157L-158R,159R-160L, 160L-161R, 162R-163R, 163R-164R, 164R-165R, 165R-166R,166R-167R, 167R-168R, 170R-171R, 173R-174R, 175R-176R, 176R-177R,178R-179R, 179R-180R, 180R-181R, 183R-184R, 184R-185L, 185L-186R,186R-187R, 187R-188R, 188R-189R, 189R-190R, and 192R-193R; and whereinone or more of the human immunodeficiency virus DNA coding sequences isan HIV vif, vpr, vpu, tat, rev, nef, gag, or pol sequence.
 2. Therecombinant MVA virus of claim 1, wherein one or more of the HIV DNAcoding sequences is a vif sequence.
 3. The recombinant MVA virus ofclaim 1, wherein one or more of the HIV DNA coding sequences is a vprsequence.
 4. The recombinant MVA virus of claim 1, wherein one or moreof the HIV DNA coding sequences is a vpu sequence.
 5. The recombinantMVA virus of claim 1, wherein one or more of the HIV DNA codingsequences is a tat sequence.
 6. The recombinant MVA virus of claim 1,wherein the tat sequence is a transdominant tat sequence.
 7. Therecombinant MVA virus of claim 1, wherein one or more of the HIV DNAcoding sequences is a rev sequence.
 8. The recombinant MVA virus ofclaim 1, wherein one or more of the HIV DNA coding sequences is a nefsequence.
 9. The recombinant MVA virus of claim 1, wherein the nefsequence is a truncated nef sequence.
 10. The recombinant MVA virus ofclaim 1, wherein one or more of the HIV DNA coding sequences is a gagsequence.
 11. The recombinant MVA virus of claim 1, wherein one or moreof the HIV DNA coding sequences is a pol sequence.
 12. The recombinantMVA virus of claim 1, comprising one or more HIV vif, vpr, vpu, tat,rev, nef, qaq, or pol vif, DNA coding sequence inserted into IGR007L-008L.
 13. The recombinant MVA virus of claim 12, wherein an HIV DNAcoding sequence encoding a Vif protein is inserted into IGR 007R-008L.14. The recombinant MVA virus of claim 12, wherein an HIV DNA codingsequence encoding a Vpr protein is inserted into IGR 007R-008L.
 15. Therecombinant MVA virus of claim 12, wherein an HIV DNA coding sequenceencoding a Vpu protein is inserted into IGR 007R-008L.
 16. Therecombinant MVA virus of claim 12, wherein an HIV DNA coding sequenceencoding a Rev protein is inserted into IGR 007R-008L.
 17. Therecombinant MVA virus of claim 1, wherein an HIV DNA coding sequenceencodes Vif-Vpr-Vpu-Rev fusion protein.
 18. The recombinant MVA virus ofclaim 17, wherein the HIV DNA coding sequence encoding theVif-Vpr-Vpu-Rev fusion protein is inserted into IGR 007R-008L.
 19. Therecombinant MVA virus of claim 1, comprising one or more HIV vif, vpr,vpu, tat, rev, nef, qaq, or pol DNA coding sequence inserted into IGR064L-065L.
 20. The recombinant MVA virus of claim 19, wherein an HIV DNAcoding sequence encoding a Nef protein is inserted into IGR 064L-065L.21. The recombinant MVA virus of claim 20, wherein the Nef protein is atruncated Nef protein.
 22. The recombinant MVA virus of claim 18,wherein an HIV DNA coding sequence encoding a truncated Nef protein isinserted into IGR 064L-065L.
 23. The recombinant MVA virus of claim 1,further comprising one or more HIV vif, vpr, vpu, tat, rev, nef, qaq, orpol DNA coding sequence inserted into IGR 136L-137L.
 24. The recombinantMVA virus of claim 23, wherein an HIV DNA coding sequence encoding a Tatprotein is inserted into IGR 136L-137L.
 25. The recombinant MVA virus ofclaim 24, the Tat protein is a transdominant Tat protein.
 26. Therecombinant MVA virus of claim 23, wherein an HIV DNA coding sequenceencoding a Gag protein is inserted into IGR 136L-137L.
 27. Therecombinant MVA virus of claim 23, wherein an HIV DNA coding sequenceencoding a Pol protein is inserted into IGR 136L-137L.
 28. Therecombinant MVA virus of claim 1, wherein an HIV DNA coding sequenceencodes encode a Gag/Pol fusion protein.
 29. The recombinant MVA virusof claim 28, wherein the HIV DNA coding sequence encoding the Gag/Polfusion protein is inserted into IGR 136L-137L.
 30. The recombinant MVAvirus of claim 22, wherein an HIV DNA coding sequence encoding a Gag/Polfusion protein is inserted into IGR 136L-137L.
 31. The recombinant MVAvirus of claim 30, wherein the HIV DNA coding sequence encoding a Tatprotein is, additionally, inserted into IGR 136L-137L.
 32. Therecombinant MVA virus of claim 31, wherein the Tat protein is truncated.